Hyperglycosylated binding polypeptides

ABSTRACT

Provided are binding polypeptides (e.g., antibodies), and effector moiety conjugates thereof, comprising a CH1 domain (e.g., a human IgG1 CH1 domain), wherein the CH1 domain has an engineered N-linked glycosylation site at amino acid position 114, according to Kabat numbering. Also provided are nucleic acids encoding the antigen-binding polypeptides, recombinant expression vectors and host cells for making such antigen-binding polypeptides. Methods of using the antigen-binding polypeptides disclosed herein to treat disease are also provided.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.14/203,438, filed Mar. 10, 2014, now U.S. Pat. No. 9,701,753, whichclaims priority to U.S. Provisional Patent Application Ser.Nos.61/776,724, 61/776,710, and 61/776,715, each filed Mar 11,2013. Thecontents of the aforementioned applications are hereby incorporated byreference herein in their entireties.

BACKGROUND

The conjugation of drugs to monoclonal antibodies or other polypeptidesoften results in the formation of a heterogeneous mixture of antibodydrug conjugates (ADCs) bearing varying number of molecules perpolypeptide. The capacity to attach molecules reliably and efficientlyto one or more defined site(s) of the antibody is highly desirable inorder to prepare chemically homogeneous compounds. Accordingly, there isa need in the art for new engineered binding polypeptides which allowfor stoichiometrically constant conjugation of moieties.

SUMMARY

The current disclosure provides binding polypeptides (e.g., antibodies),and effector moiety conjugates thereof, comprising a CH1 domain (e.g., ahuman IgG1 CH1 domain), wherein the CH1 domain has an engineeredN-linked glycosylation site at amino acid position 114, according toKabat numbering. The current disclosure also provides nucleic acidsencoding the antigen-binding polypeptides, recombinant expressionvectors and host cells for making such antigen-binding polypeptides.Methods of using the antigen-binding polypeptides disclosed herein totreat disease are also provided.

The binding polypeptides (e.g., antibodies) of the current disclosureare advantageous in that the engineered N-linked glycosylation site atKabat position 114 can also be as a site for conjugation of effectormoieties (e.g., drug moieties and targeting moieties), withoutinterfering with the antigen binding properties of the bindingpolypeptides.

Accordingly, in one aspect the disclosure provides an isolated bindingpolypeptide comprising a CH1 domain, wherein the CH1 domain comprises anasparagine residue at amino acid position 114, according to Kabatnumbering. In one embodiment, the CH1 domain further comprises: anyamino acid residue except proline at position 115, according to Kabatnumbering; and a serine or threonine residue at position 116, accordingto Kabat numbering. In one embodiment, the CH1 domain is an IgG1 CH1domain, or variant thereof. In another embodiment, the CH1 domain is ahuman IgG1 CH1 domain, or variant thereof.

In another embodiment, the side chain of the asparagine residue islinked to a glycan though a β-glycosylamide linkage. In anotherembodiment, the glycan is a biantennary glycan. In another embodiment,the glycan is a naturally occurring mammalian glycoform. In anotherembodiment, the glycan comprises a reactive aldehyde group. In anotherembodiment, the glycan comprises an oxidized saccharide residuecomprising a reactive aldehyde group. In another embodiment, theoxidized saccharide residue is a terminal sialic acid or galactose.

In another embodiment, the glycan is linked to an effector moiety. Inanother embodiment, the effector moiety is a cytotoxin. In anotherembodiment, the cytotoxin is selected from the group consisting of thecytotoxins listed in Table 1. In another embodiment, the effector moietyis a detection agent. In certain embodiments, the effector moiety is atargeting moiety. In one embodiment, the targeting moiety is acarbohydrate or glycopeptide. In another embodiment, the targetingmoiety is a glycan.

In another embodiment, the effector moiety is linked through an oxime orhydrazone linkage to saccharide residue of the glycan. In anotherembodiment, the saccharide is a terminal sialic acid or galactoseresidue of the glycan. In another embodiment, the effector moietycomprises a pH-sensitive linker, disulfide linker, enzyme-sensitivelinker or other cleavable linker moiety. In another embodiment, theeffector moiety comprises a linker moiety selected from the group oflinker moieties depicted in Table 2 or 14.

In another aspect, the invention provides an isolated bindingpolypeptide comprising a CH1 domain, wherein the CH1 domain comprises afree asparagine residue at amino acid position 114, according to Kabatnumbering.

In another aspect, the invention provides an isolated bindingpolypeptide comprising a CH1 domain, wherein the CH1 domain comprises amodified asparagine residue at amino acid position 114, according toKabat numbering, wherein said asparagine residue is linked to aneffector moiety. In one embodiment, the effector moiety is linkedthrough a side chain of the modified asparagine residue to a saccharideresidue of a glycan. In another embodiment, the saccharide is a terminalsialic acid or galactose residue of the glycan. In another embodiment,the effector moiety is linked through an oxime or hydrazone linkage tosaccharide residue of the glycan. In another embodiment, the saccharideis a terminal sialic acid or galactose residue of the glycan.

In certain embodiments, the binding polypeptide is an antibody, orfragment or derivative thereof. In another embodiment, the antibody,antibody fragment or derivative is linked to a drug effector moiety toform an antibody drug conjugate (ADC).

In certain aspects, the invention provides a composition comprising abinding polypeptide of the invention and a pharmaceutically acceptablecarrier or excipient.

In other aspects, the invention provides a method of treating a patientin thereof comprising administering an effective amount of thecomposition of the invention.

In another aspect, the invention provides an isolated polynucleotideencoding a binding polypeptide of the invention.

In another aspect, the invention provides a vector comprising thepolynucleotide. In another aspect, the invention provides a host cellcomprising the polynucleotide. In yet another aspect, the inventionprovides a method of making a binding polypeptide comprising expressingthe polynucleotide or vector in a cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of the synthesis of an antibody drugconjugate where a toxin moiety is linked to an oxidized sialic acidresidue of the antibody glycan using an oxime linkage.

FIG. 2 is a Coomassie-blue stained gel showing the expression andpurification of glycosylation mutants.

FIG. 3 depicts the results of surface plasmon resonance experiments usedto assess the binding of αβTCR HEBE1 IgG antibody mutants to recombinanthuman FcγRIIIa (V158 & F158).

FIG. 4 depicts the results of surface plasmon resonance experiments usedto assess the binding of αβTCR HEBE1 IgG antibody mutants to recombinanthuman FcγRI.

FIG. 5 depicts the cytokine release profile from PBMCs for TNFa, GM-CSF,IFNy and IL10 in the presence of mutant anti-αβTCR antibodies (day 2).

FIG. 6 depicts the cytokine release profile from PBMCs for IL6, IL4 andIL2 in the presence of mutant anti-αβTCR antibodies (day 2).

FIG. 7 depicts the cytokine release profile from PBMCs for TNFα, GM-CSF,IFNγ and IL10 in the presence of mutant anti-αβTCR antibodies (day 4).

FIG. 8 depicts the cytokine release profile from PBMCs for IL6, IL4 andIL2 in the presence of mutant anti-αβTCR antibodies (day 4).

FIG. 9 depicts the results of experiments investigating the expressionlevel of 2C3 mutants by Western blotting and surface plasmon resonance.

FIG. 10 depicts the results of experiments investigating glycosylationof 2C3 mutants pre- and post-PNGase F treatment.

FIG. 11 depicts the results of SDS-PAGE experiments investigatingglycosylation sites on 2C3 mutants isolated from cell culture.

FIG. 12 depicts the results of surface plasmon resonance experimentsused to assess the binding of modified anti-CD52 to recombinant humanFcγRIIIa (V158). Anti-CD52 comprising S298N/Y300S mutations in the Fcdomain were used to assess the effector function of the modifiedmolecule. binding to CD52 peptide (A), binding to FcγRIIIa (V158, B),andcontrol binding to mouse FcRn (C).

FIG. 13 depicts the results of surface plasmon resonance experimentsinvestigating the Fc binding properties of 2C3 mutants.

FIG. 14 depicts the results of surface plasmon resonance experimentsinvestigating the binding of modified anti-CD52 to both FcγRIIIa(Val158) (as above) and FcγRIIIa Phe158). Anti-CD52 antibodiescomprising S298N/Y300S mutations in the Fc domain were used to assessthe effector function of the modified molecule binding to FcγRIIIa(Val158, FIG. 14A) and FcγRIIIa (Phe158, FIG. 14B).

FIG. 15 depicts the analysis of C1q binding in the S298N/Y300S mutantand the WT 2C3 control (A) and the results of an Eliza analysisconfirming equivalent coating of the wells.

FIG. 16 depicts the results of plasmon resonance experiments measuringthe binding kinetics of 2C3 mutants to CD52 peptide 741.

FIG. 17 depicts the results of plasmon resonance experiments comparingthe antigen binding affinity of WT anti-CD-52 2C3 and the A114Nhyperglycosylation mutant.

FIGS. 18A to D depicts the results of isoelectric focusing and massspectrometry charge characterization experiments to determine the glycancontent of 2C3 mutants.

FIGS. 19A to B depicts the results of concentration (Octet) and plasmonresonance experiments comparing the antigen binding affinity of WTanti-CD52 2C3 and mutants.

FIG. 20 depicts the results of SDS-PAGE experiments to demonstrate theadditional glycosylation of the anti-TEM1 A114N mutant.

FIG. 21 depicts the results of SDS-PAGE and hydrophobic interactionchromatography analysis of the A114N anti-Her2 mutant.

FIG. 22 depicts the results of SDS-PAGE experiments to demonstrate theconjugation of PEG to the 2C3 A114N mutant through an aminooxy linkage.

FIG. 23 depicts the results of LC-MS experiments to determine the glycancontents of anti-TEM1 A114N hyperglycosylation mutant.

FIG. 24 depicts the results of LC-MS experiments to determine the glycancontents of a wild-type HER2 antibody and an A114N anti-Her2hyperglycosylation mutant.

FIGS. 25A to C depicts an exemplary method for performing site-specificconjugation of an antibody according to the methods of the invention.

FIG. 26 depicts a synthesis of exemplary effector moieties of theinvention: aminooxy-Cys-MC-VC-PABC-MMAE andaminooxy-Cys-MC-VC-PABC-PEG8-Dol10.

FIGS. 27A to C depicts characterization information for a sialylatedHER2 antibody.

FIGS. 28A to D depicts characterization information for oxidizedsialylated anti-HER 2 antibody.

FIG. 29 depicts hydrophobic interaction chromatographs ofglycoconjugates prepared with three different sialylated antibodies withtwo different aminooxy groups.

FIG. 30 shows a HIC chromatograph of a sialylated antibody successfullyconjugated to an aminooxy-derivatized MMAE toxin (AO-MMAE) via anengineered glycan present at the engineered glycosylation site A114N.

FIGS. 31A to D depicts a comparison of the in vitro potency of ananti-HER2 glycoconjugate and thiol conjugate.

FIG. 32 depicts a comparison of the in vitro potency of an anti FAP B11glycoconjugate and thiol conjugate.

FIG. 33 depicts a comparison of in vivo efficacy of anti-HER2glycoconjugates and thiol conjugates in a Her2+ tumor cell xenograftmodel.

FIG. 34 depicts the results of LC-MS experiments to determine the glycancontent of a mutant anti-αβTCR antibody containing the S298N/Y300Smutation.

FIG. 35 depicts the results of circular dichroism experiments todetermine the relative thermal stability of a wild-type anti-αβTCRantibody and mutant anti-αβTCR antibody containing the S298N/Y300Smutation.

DETAILED DESCRIPTION

The current disclosure provides binding polypeptides (e.g., antibodies),and effector moiety conjugates thereof, comprising a CH1 domain (e.g., ahuman IgG1 CH1 domain), wherein the CH1 domain has an engineeredN-linked glycosylation site at amino acid position 114, according toKabat numbering. The current disclosure also provides nucleic acidsencoding the antigen-binding polypeptides, recombinant expressionvectors, and host cells for making such antigen-binding polypeptides.Methods of using the antigen-binding polypeptides disclosed herein totreat disease are also provided.

I. Definitions

As used herein, the term “binding protein” or “binding polypeptide”shall refer to a polypeptide (e.g., an antibody) that contains at leastone binding site which is responsible for selectively binding to atarget antigen of interest (e.g. a human antigen). Exemplary bindingsites include an antibody variable domain, a ligand binding site of areceptor, or a receptor binding site of a ligand. In certain aspects,the binding polypeptides of the invention comprise multiple (e.g., two,three, four, or more) binding sites.

As used herein, the term “native residue” shall refer to an amino acidresidue that occurs naturally at a particular amino acid position of abinding polypeptide (e.g., an antibody or fragment thereof) and whichhas not been modified, introduced, or altered by the hand of man. Asused herein, the term “altered binding protein” or “altered bindingpolypeptide” includes binding polypeptides (e.g., an antibody orfragment thereof) comprising at least one non-native mutated amino acidresidue.

The term “specifically binds” as used herein, refers to the ability ofan antibody or an antigen-binding fragment thereof to bind to an antigenwith a dissociation constant (Kd) of at most about 1×10⁻⁶M, 1×10⁻⁷ M,1×10⁻⁸ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹M, 1×10⁻¹² M, or less, and/or tobind to an antigen with an affinity that is at least two-fold greaterthan its affinity for a nonspecific antigen.

As used herein, the term “antibody” refers to such assemblies (e.g.,intact antibody molecules, antibody fragments, or variants thereof)which have significant known specific immunoreactive activity to anantigen of interest (e.g. a tumor associated antigen). Antibodies andimmunoglobulins comprise light and heavy chains, with or without aninterchain covalent linkage between them. Basic immunoglobulinstructures in vertebrate systems are relatively well understood.

As will be discussed in more detail below, the generic term “antibody”comprises five distinct classes of antibody that can be distinguishedbiochemically. All five classes of antibodies are clearly within thescope of the current disclosure, the following discussion will generallybe directed to the IgG class of immunoglobulin molecules. With regard toIgG, immunoglobulins comprise two identical light chains of molecularweight approximately 23,000 Daltons, and two identical heavy chains ofmolecular weight 53,000-70,000. The four chains are joined by disulfidebonds in a “Y” configuration wherein the light chains bracket the heavychains starting at the mouth of the “Y” and continuing through thevariable region.

Light chains of immunoglobulin are classified as either kappa or lambda(κ, λ) Each heavy chain class may be bound with either a kappa or lambdalight chain. In general, the light and heavy chains are covalentlybonded to each other, and the “tail” portions of the two heavy chainsare bonded to each other by covalent disulfide linkages or non-covalentlinkages when the immunoglobulins are generated either by hybridomas, Bcells, or genetically engineered host cells. In the heavy chain, theamino acid sequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain. Thoseskilled in the art will appreciate that heavy chains are classified asgamma, mu, alpha, delta, or epsilon, (γ, μ, α, δ, ε) with somesubclasses among them (e.g., γ1-γ4). It is the nature of this chain thatdetermines the “class” of the antibody as IgG, IgM, IgA IgG, or IgE,respectively. The immunoglobulin isotype subclasses (e.g., IgG1, IgG2,IgG3, IgG4, IgA1, etc.) are well characterized and are known to conferfunctional specialization. Modified versions of each of these classesand isotypes are readily discernable to the skilled artisan in view ofthe instant disclosure and, accordingly, are within the scope of thecurrent disclosure.

Both the light and heavy chains are divided into regions of structuraland functional homology. The term “region” refers to a part or portionof an immunoglobulin or antibody chain and includes constant region orvariable regions, as well as more discrete parts or portions of saidregions. For example, light chain variable regions include“complementarity determining regions” or “CDRs” interspersed among“framework regions” or “FRs”, as defined herein.

The regions of an immunoglobulin heavy or light chain may be defined as“constant” (C) region or “variable” (V) regions, based on the relativelack of sequence variation within the regions of various class membersin the case of a “constant region”, or the significant variation withinthe regions of various class members in the case of a “variableregions”. The terms “constant region” and “variable region” may also beused functionally. In this regard, it will be appreciated that thevariable regions of an immunoglobulin or antibody determine antigenrecognition and specificity. Conversely, the constant regions of animmunoglobulin or antibody confer important effector functions such assecretion, transplacental mobility, Fc receptor binding, complementbinding, and the like. The subunit structures and three dimensionalconfigurations of the constant regions of the various immunoglobulinclasses are well known.

The constant and variable regions of immunoglobulin heavy and lightchains are folded into domains. The term “domain” refers to a globularregion of a heavy or light chain comprising peptide loops (e.g.,comprising 3 to 4 peptide loops) stabilized, for example, by β-pleatedsheet and/or intrachain disulfide bond. Constant region domains on thelight chain of an immunoglobulin are referred to interchangeably as“light chain constant region domains”, “CL regions” or “CL domains”.Constant domains on the heavy chain (e.g. hinge, CH1, CH2 or CH3domains) are referred to interchangeably as “heavy chain constant regiondomains”, “CH” region domains or “CH domains”. Variable domains on thelight chain are referred to interchangeably as “light chain variableregion domains”, “VL region domains or “VL domains”. Variable domains onthe heavy chain are referred to interchangeably as “heavy chain variableregion domains”, “VH region domains” or “VH domains”.

By convention the numbering of the variable constant region domainsincreases as they become more distal from the antigen binding site oramino-terminus of the immunoglobulin or antibody. The N-terminus of eachheavy and light immunoglobulin chain is a variable region and at theC-terminus is a constant region; the CH3 and CL domains actuallycomprise the carboxy-terminus of the heavy and light chain,respectively. Accordingly, the domains of a light chain immunoglobulinare arranged in a VL-CL orientation, while the domains of the heavychain are arranged in the VH—CH1-hinge-CH2-CH3 orientation.

Amino acid positions in a heavy chain constant region, including aminoacid positions in the CH1, hinge, CH2, CH3, and CL domains, may benumbered according to the Kabat index numbering system (see Kabat et al,in “Sequences of Proteins of Immunological Interest”, U.S. Dept. Healthand Human Services, 5th edition, 1991). Alternatively, antibody aminoacid positions may be numbered according to the EU index numberingsystem (see Kabat et al, ibid).

As used herein, the term “VH domain” includes the amino terminalvariable domain of an immunoglobulin heavy chain, and the term “VLdomain” includes the amino terminal variable domain of an immunoglobulinlight chain.

As used herein, the term “CH1 domain” includes the first (most aminoterminal) constant region domain of an immunoglobulin heavy chain thatextends, e.g., from about positions 114-223 in the Kabat numberingsystem (EU positions 118-215). The CH1 domain is adjacent to the VHdomain and amino terminal to the hinge region of an immunoglobulin heavychain molecule, and does not form a part of the Fc region of animmunoglobulin heavy chain.

As used herein, the term “hinge region” includes the portion of a heavychain molecule that joins the CH1 domain to the CH2 domain. This hingeregion comprises approximately 25 residues and is flexible, thusallowing the two N-terminal antigen binding regions to moveindependently. Hinge regions can be subdivided into three distinctdomains: upper, middle, and lower hinge domains (Roux et al. J. Immunol.1998, 161:4083).

As used herein, the term “CH2 domain” includes the portion of a heavychain immunoglobulin molecule that extends, e.g., from about positions244-360 in the Kabat numbering system (EU positions 231-340). The CH2domain is unique in that it is not closely paired with another domain.Rather, two N-linked branched carbohydrate chains are interposed betweenthe two CH2 domains of an intact native IgG molecule. In one embodiment,a binding polypeptide of the current disclosure comprises a CH2 domainderived from an IgG1 molecule (e.g. a human IgG1 molecule).

As used herein, the term “CH3 domain” includes the portion of a heavychain immunoglobulin molecule that extends approximately 110 residuesfrom N-terminus of the CH2 domain, e.g., from about positions 361-476 ofthe Kabat numbering system (EU positions 341-445). The CH3 domaintypically forms the C-terminal portion of the antibody. In someimmunoglobulins, however, additional domains may extend from CH3 domainto form the C-terminal portion of the molecule (e.g. the CH4 domain inthe μ chain of IgM and the e chain of IgE). In one embodiment, a bindingpolypeptide of the current disclosure comprises a CH3 domain derivedfrom an IgG1 molecule (e.g. a human IgG1 molecule).

As used herein, the term “CL domain” includes the constant region domainof an immunoglobulin light chain that extends, e.g. from about Kabatposition 107A-216. The CL domain is adjacent to the VL domain. In oneembodiment, a binding polypeptide of the current disclosure comprises aCL domain derived from a kappa light chain (e.g., a human kappa lightchain).

As used herein, the term “Fc region” is defined as the portion of aheavy chain constant region beginning in the hinge region just upstreamof the papain cleavage site (i.e. residue 216 in IgG, taking the firstresidue of heavy chain constant region to be 114) and ending at theC-terminus of the antibody. Accordingly, a complete Fc region comprisesat least a hinge domain, a CH2 domain, and a CH3 domain.

The term “native Fc” as used herein refers to a molecule comprising thesequence of a non-antigen-binding fragment resulting from digestion ofan antibody or produced by other means, whether in monomeric ormultimeric form, and can contain the hinge region. The originalimmunoglobulin source of the native Fc is preferably of human origin andcan be any of the immunoglobulins, although IgG1 and IgG2 are preferred.Native Fc molecules are made up of monomeric polypeptides that can belinked into dimeric or multimeric forms by covalent (i.e., disulfidebonds) and non-covalent association. The number of intermoleculardisulfide bonds between monomeric subunits of native Fc molecules rangesfrom 1 to 4 depending on class (e.g., IgG, IgA, and IgE) or subclass(e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). One example of a native Fc isa disulfide-bonded dimer resulting from papain digestion of an IgG. Theterm “native Fc” as used herein is generic to the monomeric, dimeric,and multimeric forms.

The term “Fc variant” as used herein refers to a molecule or sequencethat is modified from a native Fc but still comprises a binding site forthe salvage receptor, FcRn (neonatal Fc receptor). Exemplary Fcvariants, and their interaction with the salvage receptor, are known inthe art. Thus, the term “Fc variant” can comprise a molecule or sequencethat is humanized from a non-human native Fc. Furthermore, a native Fccomprises regions that can be removed because they provide structuralfeatures or biological activity that are not required for theantibody-like binding polypeptides of the invention. Thus, the term “Fcvariant” comprises a molecule or sequence that lacks one or more nativeFc sites or residues, or in which one or more Fc sites or residues hasbe modified, that affect or are involved in: (1) disulfide bondformation, (2) incompatibility with a selected host cell, (3)N-terminalheterogeneity upon expression in a selected host cell, (4)glycosylation, (5) interaction with complement, (6) binding to an Fcreceptor other than a salvage receptor, or (7) antibody-dependentcellular cytotoxicity (ADCC).

The term “Fc domain” as used herein encompasses native Fc and Fcvariants and sequences as defined above. As with Fc variants and nativeFc molecules, the term “Fc domain” includes molecules in monomeric ormultimeric form, whether digested from whole antibody or produced byother means.

As indicated above, the variable regions of an antibody allow it toselectively recognize and specifically bind epitopes on antigens. Thatis, the VL domain and VH domain of an antibody combine to form thevariable region (Fv) that defines a three dimensional antigen bindingsite. This quaternary antibody structure forms the antigen binding sitepresent at the end of each arm of the Y. More specifically, the antigenbinding site is defined by three complementary determining regions(CDRs) on each of the heavy and light chain variable regions. As usedherein, the term “antigen binding site” includes a site thatspecifically binds (immunoreacts with) an antigen (e.g., a cell surfaceor soluble antigen). The antigen binding site includes an immunoglobulinheavy chain and light chain variable region and the binding site formedby these variable regions determines the specificity of the antibody. Anantigen binding site is formed by variable regions that vary from oneantibody to another. The altered antibodies of the current disclosurecomprise at least one antigen binding site.

In certain embodiments, binding polypeptides of the current disclosurecomprise at least two antigen binding domains that provide for theassociation of the binding polypeptide with the selected antigen. Theantigen binding domains need not be derived from the same immunoglobulinmolecule. In this regard, the variable region may or be derived from anytype of animal that can be induced to mount a humoral response andgenerate immunoglobulins against the desired antigen. As such, thevariable region of the a binding polypeptide may be, for example, ofmammalian origin e.g., may be human, murine, rat, goat, sheep, non-humanprimate (such as cynomolgus monkeys, macaques, etc.), lupine, or camelid(e.g., from camels, llamas and related species).

In naturally occurring antibodies, the six CDRs present on eachmonomeric antibody are short, non-contiguous sequences of amino acidsthat are specifically positioned to form the antigen binding site as theantibody assumes its three dimensional configuration in an aqueousenvironment. The remainder of the heavy and light variable domains showless inter-molecular variability in amino acid sequence and are termedthe framework regions. The framework regions largely adopt a β-sheetconformation and the CDRs form loops which connect, and in some casesform part of, the β-sheet structure. Thus, these framework regions actto form a scaffold that provides for positioning the six CDRs in correctorientation by inter-chain, non-covalent interactions. The antigenbinding domain formed by the positioned CDRs defines a surfacecomplementary to the epitope on the immunoreactive antigen. Thiscomplementary surface promotes the non-covalent binding of the antibodyto the immunoreactive antigen epitope.

Exemplary binding polypeptides of the invention include antibodyvariants. As used herein, the term “antibody variant” includes syntheticand engineered forms of antibodies which are altered such that they arenot naturally occurring, e.g., antibodies that comprise at least twoheavy chain portions but not two complete heavy chains (such as, domaindeleted antibodies or minibodies); multispecific forms of antibodies(e.g., bispecific, trispecific, etc.) altered to bind to two or moredifferent antigens or to different epitopes on a single antigen); heavychain molecules joined to scFv molecules and the like. In addition, theterm “antibody variant” includes multivalent forms of antibodies (e.g.,trivalent, tetravalent, etc., antibodies that bind to three, four ormore copies of the same antigen.

As used herein the term “valency” refers to the number of potentialtarget binding sites in a polypeptide. Each target binding sitespecifically binds one target molecule or specific site on a targetmolecule. When a polypeptide comprises more than one target bindingsite, each target binding site may specifically bind the same ordifferent molecules (e.g., may bind to different ligands or differentantigens, or different epitopes on the same antigen). The subjectbinding polypeptides preferably have at least one binding site specificfor a human antigen molecule.

The term “specificity” refers to the ability to specifically bind (e.g.,immunoreact with) a given target antigen (e.g., a human target antigen).A binding polypeptide may be monospecific and contain one or morebinding sites which specifically bind a target or a polypeptide may bemultispecific and contain two or more binding sites which specificallybind the same or different targets. In certain embodiments, a bindingpolypeptide of the invention is specific for two different (e.g.,non-overlapping) portions of the same target. In certain embodiments, abinding polypeptide of the invention is specific for more than onetarget. Exemplary binding polypeptides (e.g., antibodies) which compriseantigen binding sites that bind to antigens expressed on tumor cells areknown in the art and one or more CDRs from such antibodies can beincluded in an antibody of the invention.

The term “linking moiety” includes moieties which are capable of linkingthe effector moiety to the binding polypeptides disclosed herein. Thelinking moiety may be selected such that it is cleavable (e.g.,enzymatically cleavable or pH-sensitive) or non-cleavable. Exemplarylinking moieties are set forth in Table 2 herein.

As used herein, the term “effector moiety” comprises agents (e.g.proteins, nucleic acids, lipids, carbohydrates, glycopeptides, drugmoieties, and fragments thereof) with biological or other functionalactivity. For example, a modified binding polypeptide comprising aneffector moiety conjugated to a binding polypeptide has at least oneadditional function or property as compared to the unconjugatedantibody. For example, the conjugation of a cytotoxic drug (e.g., aneffector moiety) to binding polypeptide results in the formation of abinding polypeptide with drug cytotoxicity as second function (i.e. inaddition to antigen binding). In another example, the conjugation of asecond binding polypeptide to the binding polypeptide may conferadditional binding properties. In certain embodiments, where theeffector moiety is a genetically encoded therapeutic or diagnosticprotein or nucleic acid, the effector moiety may be synthesized orexpressed by either peptide synthesis or recombinant DNA methods thatare well known in the art. In another aspect, where the effector moietyis a non-genetically encoded peptide, or a drug moiety, the effectormoiety may be synthesized artificially or purified from a naturalsource. As used herein, the term “drug moiety” includesanti-inflammatory, anticancer, anti-infective (e.g., anti-fungal,antibacterial, anti-parasitic, anti-viral, etc.), and anesthetictherapeutic agents. In a further embodiment, the drug moiety is ananticancer or cytotoxic agent. Compatible drug moieties may alsocomprise prodrugs. Exemplary effector moieties are set forth in Table 1herein.

In certain embodiments, an “effector moiety” comprises a “targetingmoiety.” As used herein, the term “targeting moiety” refers to aneffector moiety that binds to a target molecule. Targeting moieties cancomprise, without limitation, proteins, nucleic acids, lipids,carbohydrates (e.g., glycans), and combinations thereof (e.g.,glycoproteins, glycopeptides, and glycolipids).

As used herein, the term “prodrug” refers to a precursor or derivativeform of a pharmaceutically active agent that is less active, reactive orprone to side effects as compared to the parent drug and is capable ofbeing enzymatically activated or otherwise converted into a more activeform in vivo. Prodrugs compatible with the compositions of the currentdisclosure include, but are not limited to, phosphate-containingprodrugs, amino acid-containing prodrugs, thiophosphate-containingprodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,β-lactam-containing prodrugs, optionally substitutedphenoxyacetamide-containing prodrugs or optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs that can be converted to the more activecytotoxic free drug. One skilled in the art may make chemicalmodifications to the desired drug moiety or its prodrug in order to makereactions of that compound more convenient for purposes of preparingmodified binding polypeptides of the current disclosure. The drugmoieties also include derivatives, pharmaceutically acceptable salts,esters, amides, and ethers of the drug moieties described herein.Derivatives include modifications to drugs identified herein which mayimprove or not significantly reduce a particular drug's desiredtherapeutic activity.

As used herein, the term “anticancer agent” includes agents which aredetrimental to the growth and/or proliferation of neoplastic or tumorcells and may act to reduce, inhibit or destroy malignancy. Examples ofsuch agents include, but are not limited to, cytostatic agents,alkylating agents, antibiotics, cytotoxic nucleosides, tubulin bindingagents, hormones, hormone antagonists, cytotoxic agents, and the like.Cytotoxic agents include tomaymycin derivatives, maytansine derivatives,cryptophycine derivatives, anthracycline derivatives, bisphosphonatederivatives, leptomycin derivatives, streptonigrin derivatives,auristatine derivatives, and duocarmycin derivatives. Any agent thatacts to retard or slow the growth of immunoreactive cells or malignantcells is within the scope of the current disclosure.

The term “antigen” or “target antigen” as used herein refers to amolecule or a portion of a molecule that is capable of being bound bythe binding site of a binding polypetpide. A target antigen may have oneor more epitopes.

II. Binding Polypeptides

In one aspect, the current disclosure provides binding polypeptides(e.g., antibodies, antibody fragments, antibody variants, and fusionproteins) comprising at least one CH1 domain having an N-linkedglycosylation site at position 114, according to Kabat numbering.

CH1 domains from any immunoglobulin class (e.g., IgM, IgG, IgD, IgA andIgE) and species can be used in the binding polypeptides disclosedherein. Chimeric CHI domains comprising portions of CHI domains fromdifferent species or Ig classes can also be employed. In certainembodiments, the CH1 domain is a human IgG1 CH1 domain. In the case of ahuman IgG1 domain, mutation of the wild type amino acid at position 114to an asparagine results in the formation of an N-linked glycosylationconsensus site (i.e, the N-X-T/S sequon, where X is any amino acidexcept proline). However, in the case of other CH1 domains of otherspecies and/or Ig classes or isotypes, the skilled artisan willappreciate that it may be necessary to mutate positions 115 and/or 116of the CH1 domain to create an N-X-T/S sequon.

The binding polypeptides disclosed herein encompass any bindingpolypeptide that comprises a CH1 domain having an N-linked glycosylationsite at position 114, according to Kabat numbering.

In certain embodiments, the binding polypeptide is an antibody, orfragment or derivative thereof. Any antibody from any source or speciescan be employed in the binding polypeptides disclosed herein. Suitableantibodies include without limitation, human antibodies, humanizedantibodies or chimeric antibodies.

In certain embodiments, the binding polypeptide of the currentdisclosure may comprise an antigen binding fragment of an antibody. Theterm “antigen-binding fragment” refers to a polypeptide fragment of animmunoglobulin or antibody which binds antigen or competes with intactantibody {i.e., with the intact antibody from which they were derived)for antigen binding (i.e., specific binding). Antigen binding fragmentscan be produced by recombinant or biochemical methods that are wellknown in the art. Exemplary antigen-binding fragments include Fv, Fab,Fab′, and (Fab′)2. In preferred embodiments, the antigen-bindingfragment of the current disclosure is an altered antigen-bindingfragment comprising at least one engineered glycosylation site. In oneexemplary embodiment, an altered antigen binding fragment of the currentdisclosure comprises an altered VH domain described supra. In anotherexemplary embodiment, an altered antigen binding fragment of the currentdisclosure comprises an altered CH1 domain described supra.

In exemplary embodiments, the binding polypeptide comprises a singlechain variable region sequence (ScFv). Single chain variable regionsequences comprise a single polypeptide having one or more antigenbinding sites, e.g., a VL domain linked by a flexible linker to a VHdomain. ScFv molecules can be constructed in a VH-linker-VL orientationor VL-linker-VH orientation. The flexible hinge that links the VL and VHdomains that make up the antigen binding site preferably comprises fromabout 10 to about 50 amino acid residues. Connecting peptides are knownin the art. Binding polypeptide of the invention may comprise at leastone scFv and/or at least one constant region. In one embodiment, abinding polypeptide of the current disclosure may comprise at least onescFv linked or fused to an antibody or fragment comprising a CH1 domain(e.g. a CH1 domain comprising an asparagine residue at Kabat position114) and/or a CH2 domain (e.g. a CH2 domain comprising an asparagineresidue at EU position 298, and a serine or threonine residue at EUposition 300).

In certain exemplary embodiments, a binding polypeptide of the currentdisclosure is a multivalent (e.g., tetravalent) antibody which isproduced by fusing a DNA sequence encoding an antibody with a ScFvmolecule (e.g., an altered ScFv molecule). For example, in oneembodiment, these sequences are combined such that the ScFv molecule(e.g., an altered ScFv molecule) is linked at its N-terminus orC-terminus to an Fc fragment of an antibody via a flexible linker (e.g.,a gly/ser linker). In another embodiment a tetravalent antibody of thecurrent disclosure can be made by fusing an ScFv molecule to aconnecting peptide, which is fused to a CH1 domain (e.g. a CH1 domaincomprising an asparagine residue at Kabat position 114) to construct anScFv-Fab tetravalent molecule.

In another embodiment, a binding polypeptide of the current disclosureis an altered minibody. Altered minibodies of the current disclosure aredimeric molecules made up of two polypeptide chains each comprising anScFv molecule (e.g., an altered ScFv molecule comprising an altered VHdomain described supra) which is fused to a CH3 domain or portionthereof via a connecting peptide. Minibodies can be made by constructingan ScFv component and connecting peptide-CH3 components using methodsdescribed in the art (see, e.g., U.S. Pat. No. 5,837,821 orWO94/09817A1). In another embodiment, a tetravalent minibody can beconstructed. Tetravalent minibodies can be constructed in the samemanner as minibodies, except that two ScFv molecules are linked using aflexible linker. The linked scFv-scFv construct is then joined to a CH3domain.

In another embodiment, a binding polypeptide of the current disclosurecomprises a diabody. Diabodies are dimeric, tetravalent molecules eachhaving a polypeptide similar to scFv molecules, but usually having ashort (less than 10 and preferably 1-5) amino acid residue linkerconnecting both variable domains, such that the VL and VH domains on thesame polypeptide chain cannot interact. Instead, the VL and VH domain ofone polypeptide chain interact with the VH and VL domain (respectively)on a second polypeptide chain (see, for example, WO02/02781). Diabodiesof the current disclosure comprise an scFv molecule fused to a CH3domain.

In other embodiments, the binding polypeptide s of the inventioncomprise multispecific or multivalent antibodies comprising one or morevariable domain in series on the same polypeptide chain, e.g., tandemvariable domain (TVD) polypeptides. Exemplary TVD polypeptides includethe “double head” or “Dual-Fv” configuration described in U.S. Pat. No.5,989,830. In the Dual-Fv configuration, the variable domains of twodifferent antibodies are expressed in a tandem orientation on twoseparate chains (one heavy chain and one light chain), wherein onepolypeptide chain has two VH domains in series separated by a peptidelinker (VH1-linker-VH2) and the other polypeptide chain consists ofcomplementary VL domains connected in series by a peptide linker(VL1-linker-VL2). In the cross-over double head configuration, thevariable domains of two different antibodies are expressed in a tandemorientation on two separate polypeptide chains (one heavy chain and onelight chain), wherein one polypeptide chain has two VH domains in seriesseparated by a peptide linker (VH1-linker-VH2) and the other polypeptidechain consists of complementary VL domains connected in series by apeptide linker in the opposite orientation (VL2-linker-VL1). Additionalantibody variants based on the “Dual-Fv” format include theDual-Variable-Domain IgG (DVD-IgG) bispecific antibody (see U.S. Pat.No. 7,612,181 and the TBTI format (see US 2010/0226923 A1). The additionof constant domains to respective chains of the Dual-Fv (CH1-Fc to theheavy chain and kappa or lambda constant domain to the light chain)leads to functional bispecific antibodies without any need foradditional modifications (i.e., obvious addition of constant domains toenhance stability).

In another exemplary embodiment, the binding polypeptide comprises across-over dual variable domain IgG (CODV-IgG) bispecific antibody basedon a “double head” configuration (see US20120251541 A1, which isincorporated by reference herein in its entirety). CODV-IgG antibodyvariants have one polypeptide chain with VL domains connected in seriesto a CL domain (VL1-L1-VL2-L2-CL) and a second polypeptide chain withcomplementary VH domains connected in series in the opposite orientationto a CH1 domain (VH2-L3-VH1-L4-CH1), where the polypeptide chains form across-over light chain-heavy chain pair. In certain embodiment, thesecond polypeptide may be further connected to an Fc domain(VH2-L3-VH1-L4-CH1-Fc). In certain embodiments, linker L3 is at leasttwice the length of linker L1 and/or linker L4 is at least twice thelength of linker L2. For example, L1 and L2 may be 1-3 amino acidresidues in length, L3 may be 2 to 6 amino acid residues in length, andL4 may be 4 to 7 amino acid residues in length. Examples of suitablelinkers include a single glycine (Gly) residue; a diglycine peptide(Gly-Gly); a tripeptide (Gly-Gly-Gly); a peptide with four glycineresidues (Gly-Gly-Gly-Gly; SEQ ID NO: 17);a peptide with five glycineresidues (Gly-Gly-Gly-Gly-Gly; SEQ ID NO: 18); a peptide with sixglycine residues (Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO: 19); a peptidewith seven glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO:20); a peptide with eight glycine residues(Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO: 21). Other combinations ofamino acid residues may be used such as the peptide Gly-Gly-Gly-Gly-Ser(SEQ ID NO: 22) and the peptide Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser(SEQ ID NO: 39).

In certain embodiments, the binding polypeptide comprises animmunoadhesin molecule comprising a non-antibody binding region (e.g., areceptor, ligand, or cell-adhesion molecule) fused to an antibodyconstant region (see e.g., Ashkenazi et al., Methods, 1995 8(2),104-115, which is incorporated by reference herein in its entirety)

In certain embodiments, the binding polypeptide comprisesimmunoglobulin-like domains. Suitable immunoglobulin-like domainsinclude, without limitation, fibronectin domains (see, for example,Koide et al. (2007), Methods Mol. Biol. 352: 95-109, which isincorporated by reference herein in its entirety), DARPin (see, forexample, Stumpp et al. (2008) Drug Discov. Today 13 (15-16): 695-701,which is incorporated by reference herein in its entirety), Z domains ofprotein A (see, Nygren et al. (2008) FEBS J. 275 (11): 2668-76, which isincorporated by reference herein in its entirety), Lipocalins (see, forexample, Skerra et al. (2008) FEBS J. 275 (11): 2677-83, which isincorporated by reference herein in its entirety), Affilins (see, forexample, Ebersbach et al. (2007) J. Mol. Biol. 372 (1): 172-85, which isincorporated by reference herein in its entirety), Affitins (see, forexample, Krehenbrink et al. (2008). J. Mol. Biol. 383 (5): 1058-68,which is incorporated by reference herein in its entirety), Avimers(see, for example, Silverman et al. (2005) Nat. Biotechnol. 23 (12):1556-61, which is incorporated by reference herein in its entirety),Fynomers, (see, for example, Grabulovski et al. (2007) J Biol Chem 282(5): 3196-3204, which is incorporated by reference herein in itsentirety), and Kunitz domain peptides (see, for example, Nixon et al.(2006) Curr Opin Drug Discov Devel 9 (2): 261-8, which is incorporatedby reference herein in its entirety).

III. N-linked Glycans

In certain embodiments, the CH1 domain of the binding polypeptidesdisclosed herein is glycosylated at the engineered arginine at position114 (N114), according to Kabat numbering. The N-linked glycan isgenerally linked though a β-glycosylamide linkage to the nitrogen groupof the N114 side chain. However, other suitable art recognized linkagescan also be employed.

Any type of naturally occurring or synthetic (i.e., non-natural)N-linkedglycan can be linked to N114. For example, the glycan may be a nativeglycan or an engineered glycan containing non-native linkages. Incertain embodiments, the glycan comprises a saccharide that can beoxidized (e.g., by periodate treatment) to produce a group suitable forconjugation to an effector moiety (e.g., a reactive aldehyde group).Suitable oxidizable saccharides included, without limitation, galactoseand sialic acid (e.g., N-Acetylneuraminic acid). In certain embodiments,the glycan is a biantennary glycan. In certain embodiments, the glycanis a naturally occurring mammalian glycoform.

Glycosylation can be achieved through any means known in the art. Incertain embodiments, the glycosylation is achieved by expression of thebinding polypeptides in cells capable of N-linked glycosylation. Anynatural or engineered cell (e.g., prokaryotic or eukaryotic) can beemployed. In general, mammalian cells are employed to effectglycosylation. The N-glycans that are produced in mammalian cells arecommonly referred to as complex N-glycans (see e.g., Drickamer K, TaylorM E (2006). Introduction to Glycobiology, 2nd ed., which is incorporatedherein by reference in its entirety). These complex N-glycans have astructure that typically has two to six outer branches with asialyllactosamine sequence linked to an inner core structureMan₃GlcNAc₂. A complex N-glycan has at least one branch, and preferablyat least two, of alternating GlcNAc and galactose (Gal) residues thatterminate in oligosaccharides such as, for example: NeuNAc-; NeuAc α2,6GalNAc α1-; NeuAc α2,3 Gal.beta.1 3GalNAc α 1-; and NeuAc α.2,3/6 Galβ1,4 GlcNAc β1. In addition, sulfate esters can occur on galactose,GalNAc, and GlcNAc residues, and phosphate esters can occur on mannoseresidues. NeuAc can be O-acetylated or replaced by NeuG1(N-glycolylneuraminic acid). Complex N-glycans may also have intrachainsubstitutions of bisecting GlcNAc and core fucose (Fuc).

Additionally or alternatively, glycosylation can be achieved or modifiedthrough enzymatic means, in vitro. For example, one or moreglycosyltransferases may be employed to add specific saccharide residuesto N114, and one or more glycosidases may be employed to remove unwantedsaccharides from the N-linked glycan. Such enzymatic means are wellknown in the art (see. e.g., WO/2007/005786, which is incorporatedherein by reference in its entirety).

IV. Immunological Effector Functions and Fc Modifications

In certain embodiments, binding polypeptides of the invention maycomprise an antibody constant region (e.g. an IgG constant region e.g.,a human IgG constant region, e.g., a human IgG1 or IgG4 constant region)which mediates one or more effector functions. For example, binding ofthe C1-complex to an antibody constant region may activate thecomplement system. Activation of the complement system is important inthe opsonisation and lysis of cell pathogens. The activation of thecomplement system also stimulates the inflammatory response and may alsobe involved in autoimmune hypersensitivity. Further, antibodies bind toreceptors on various cells via the Fc region (Fc receptor binding siteson the antibody Fc region bind to Fc receptors (FcRs) on a cell). Thereare a number of Fc receptors which are specific for different classes ofantibody, including IgG (gamma receptors), IgE (epsilon receptors), IgA(alpha receptors) and IgM (mu receptors). Binding of antibody to Fcreceptors on cell surfaces triggers a number of important and diversebiological responses including engulfment and destruction ofantibody-coated particles, clearance of immune complexes, lysis ofantibody-coated target cells by killer cells (called antibody-dependentcell-mediated cytotoxicity, or ADCC), release of inflammatory mediators,placental transfer and control of immunoglobulin production. Inpreferred embodiments, the binding polypeptides (e.g., antibodies orantigen binding fragments thereof) of the invention bind to an Fc-gammareceptor. In alternative embodiments, binding polypeptides of theinvention may comprise a constant region which is devoid of one or moreeffector functions (e.g., ADCC activity) and/or is unable to bind Fcγreceptor.

Certain embodiments of the invention include antibodies in which atleast one amino acid in one or more of the constant region domains hasbeen deleted or otherwise altered so as to provide desired biochemicalcharacteristics such as reduced or enhanced effector functions, theability to non-covalently dimerize, increased ability to localize at thesite of a tumor, reduced serum half-life, or increased serum half-lifewhen compared with a whole, unaltered antibody of approximately the sameimmunogenicity. For example, certain antibodies for use in thediagnostic and treatment methods described herein are domain deletedantibodies which comprise a polypeptide chain similar to animmunoglobulin heavy chain, but which lack at least a portion of one ormore heavy chain domains. For instance, in certain antibodies, oneentire domain of the constant region of the modified antibody will bedeleted, for example, all or part of the CH2 domain will be deleted.

In certain other embodiments, binding polypeptides comprise constantregions derived from different antibody isotypes (e.g., constant regionsfrom two or more of a human IgG1, IgG2, IgG3, or IgG4). In otherembodiments, binding polypeptides comprises a chimeric hinge (i.e., ahinge comprising hinge portions derived from hinge domains of differentantibody isotypes, e.g., an upper hinge domain from an IgG4 molecule andan IgG1 middle hinge domain). In one embodiment, binding polypeptidescomprise an Fc region or portion thereof from a human IgG4 molecule anda Ser228Pro mutation (EU numbering) in the core hinge region of themolecule.

In certain embodiments, the Fc portion may be mutated to increase ordecrease effector function using techniques known in the art. Forexample, the deletion or inactivation (through point mutations or othermeans) of a constant region domain may reduce Fc receptor binding of thecirculating modified antibody thereby increasing tumor localization. Inother cases it may be that constant region modifications consistent withthe instant invention moderate complement binding and thus reduce theserum half life and nonspecific association of a conjugated cytotoxin.Yet other modifications of the constant region may be used to modifydisulfide linkages or oligosaccharide moieties that allow for enhancedlocalization due to increased antigen specificity or flexibility. Theresulting physiological profile, bioavailability and other biochemicaleffects of the modifications, such as tumor localization,biodistribution and serum half-life, may easily be measured andquantified using well know immunological techniques without undueexperimentation.

In certain embodiments, an Fc domain employed in an antibody of theinvention is an Fc variant. As used herein, the term “Fc variant” refersto an Fc domain having at least one amino acid substitution relative tothe wild-type Fc domain from which said Fc domain is derived. Forexample, wherein the Fc domain is derived from a human IgG1 antibody,the Fc variant of said human IgG1 Fc domain comprises at least one aminoacid substitution relative to said Fc domain.

The amino acid substitution(s) of an Fc variant may be located at anyposition (i.e., any EU convention amino acid position) within the Fcdomain. In one embodiment, the Fc variant comprises a substitution at anamino acid position located in a hinge domain or portion thereof. Inanother embodiment, the Fc variant comprises a substitution at an aminoacid position located in a CH2 domain or portion thereof. In anotherembodiment, the Fc variant comprises a substitution at an amino acidposition located in a CH3 domain or portion thereof. In anotherembodiment, the Fc variant comprises a substitution at an amino acidposition located in a CH4 domain or portion thereof.

The binding polypeptides of the invention may employ any art-recognizedFc variant which is known to impart an improvement (e.g., reduction orenhancement) in effector function and/or FcR binding. Said Fc variantsmay include, for example, any one of the amino acid substitutionsdisclosed in International PCT Publications WO88/07089A1, WO96/14339A1,WO98/05787A1, WO98/23289A1, WO99/51642A1, WO99/58572A1, WO00/09560A2,WO00/32767A1, WO00/42072A2, WO02/44215A2, WO02/060919A2, WO03/074569A2,WO04/016750A2, WO04/029207A2, WO04/035752A2, WO04/063351A2,WO04/074455A2, WO04/099249A2, WO05/040217A2, WO05/070963A1,WO05/077981A2, WO05/092925A2, WO05/123780A2, WO06/019447A1,WO06/047350A2, and WO06/085967A2 or U.S. Pat. Nos. 5,648,260; 5,739,277;5,834,250; 5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195;6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; and7,083,784, each of which is incorporated in its entirety by referenceherein. In one exemplary embodiment, a binding polypeptide of theinvention may comprise an Fc variant comprising an amino acidsubstitution at EU position 268 (e.g., H268D or H268E). In anotherexemplary embodiment, a binding polypeptide of the invention maycomprise an amino acid substitution at EU position 239 (e.g., S239D orS239E) and/or EU position 332 (e.g., I332D or I332Q).

In certain embodiments, a binding polypeptide of the invention maycomprise an Fc variant comprising an amino acid substitution whichalters the antigen-independent effector functions of the antibody, inparticular the circulating half-life of the binding polypeptide. Suchbinding polypeptides exhibit either increased or decreased binding toFcRn when compared to binding polypeptides lacking these substitutions,therefore, have an increased or decreased half-life in serum,respectively. Fc variants with improved affinity for FcRn areanticipated to have longer serum half-lives, and such molecules haveuseful applications in methods of treating mammals where long half-lifeof the administered antibody is desired, e.g., to treat a chronicdisease or disorder. In contrast, Fc variants with decreased FcRnbinding affinity are expected to have shorter half-lives, and suchmolecules are also useful, for example, for administration to a mammalwhere a shortened circulation time may be advantageous, e.g. for in vivodiagnostic imaging or in situations where the starting antibody hastoxic side effects when present in the circulation for prolongedperiods. Fc variants with decreased FcRn binding affinity are also lesslikely to cross the placenta and, thus, are also useful in the treatmentof diseases or disorders in pregnant women. In addition, otherapplications in which reduced FcRn binding affinity may be desiredinclude applications localized to the brain, kidney, and/or liver. Inone exemplary embodiment, the altered binding polypeptides (e.g.,antibodies or antigen binding fragments thereof) of the inventionexhibit reduced transport across the epithelium of kidney glomeruli fromthe vasculature. In another embodiment, the altered binding polypeptides(e.g., antibodies or antigen binding fragments thereof) of the inventionexhibit reduced transport across the blood brain bather (BBB) from thebrain, into the vascular space. In one embodiment, an antibody withaltered FcRn binding comprises an Fc domain having one or more aminoacid substitutions within the “FcRn binding loop” of an Fc domain. TheFcRn binding loop is comprised of amino acid residues 280-299 (accordingto EU numbering). Exemplary amino acid substitutions which alter FcRnbinding activity are disclosed in International PCT Publication No.WO05/047327 which is incorporated in its entirety by reference herein.In certain exemplary embodiments, the binding polypeptides (e.g.,antibodies or antigen binding fragments thereof) of the inventioncomprise an Fc domain having one or more of the following substitutions:V284E, H285E, N286D, K290E and 5304D (EU numbering). In yet otherexemplary embodiments, the biding molecules of the invention comprise ahuman Fc domain with the double mutation H433K/N434F (see, e.g., U.S.Pat. No. 8,163,881).

In other embodiments, binding polypeptides, for use in the diagnosticand treatment methods described herein have a constant region, e.g., anIgG1 or IgG4 heavy chain constant region, which is altered to reduce oreliminate glycosylation. For example, binding polypeptides (e.g.,antibodies or antigen binding fragments thereof) of the invention mayalso comprise an Fc variant comprising an amino acid substitution whichalters the glycosylation of the antibody Fc. For example, said Fcvariant may have reduced glycosylation (e.g., N- or O-linkedglycosylation). In exemplary embodiments, the Fc variant comprisesreduced glycosylation of the N-linked glycan normally found at aminoacid position 297 (EU numbering). In another embodiment, the antibodyhas an amino acid substitution near or within a glycosylation motif, forexample, an N-linked glycosylation motif that contains the amino acidsequence NXT or NXS. In a particular embodiment, the antibody comprisesan Fc variant with an amino acid substitution at amino acid position 228or 299 (EU numbering). In certain embodiments, the binding polypeptidecomprises an IgG1 or IgG4 constant region comprising an S228P and aT299A mutation (EU numbering).

In certain embodiments, the Fc domain of the binding polypeptidecomprises an Fc variant with an engineered arginine at position 298,according to EU numbering, and an engineered arginine serine orthreonine residue at amino acid position 300 (EU numbering). This Fcvariant is glycosylated ay position 298 and inhibits binding to Fcγreceptors.

Exemplary amino acid substitutions which confer reduce or alteredglycosylation are disclosed in International PCT Publication No.WO05/018572, which is incorporated in its entirety by reference herein.In preferred embodiments, the binding polypeptides of the invention aremodified to eliminate glycosylation. Such binding polypeptides may bereferred to as “agly” binding polypeptides (e.g. “agly” antibodies).While not being bound by theory, it is believed that “agly” bindingpolypeptides may have an improved safety and stability profile in vivo.Agly binding polypeptides can be of any isotype or subclass thereof,e.g., IgG1, IgG2, IgG3, or IgG4. In certain embodiments, agly bindingpolypeptides comprise an aglycosylated Fc region of an IgG4 antibodywhich is devoid of Fc-effector function. In yet other embodiments,binding polypeptides of the invention comprise an altered glycan. Forexample, the antibody may have a reduced number of fucose residues on anN-glycan at Asn297 of the Fc region, i.e., is afucosylated.Afucosylation increases FcRgII binding on the NK cells and potentlyincrease ADCC. In another embodiment, the binding polypeptide may havean altered number of sialic acid residues on the N-glycan at Asn297 ofthe Fc region. Numerous art-recognized methods are available for making“agly” antibodies or antibodies with altered glycans. For examples,genetically engineered host cells (e.g., modified yeast, e.g., Picchia,or CHO cells) with modified glycosylation pathways (e.g.,glycosyl-transferase deletions) can be used to produce such antibodies.

V. Effector Moieties

In certain embodiments, the binding polypeptides of the currentdisclosure comprise effector moieties. In general these effectormoieties are conjugated (either directly or through a linker moiety) toan N-linked glycan on the binding polypeptide, (e.g., an N-linked glycanlinked to N114 of a CH1 domain). In certain embodiments, the bindingpolypeptide is full length antibody comprising two CH1 domains with aglycan at Kabat position 114, wherein both of the glycans are conjugatedto one or more effector moieties.

Any effector moiety can be added to the binding polypeptides disclosedherein. The effector moieties preferably add a non-natural function toan altered antibody or fragments thereof, without significantly alteringthe intrinsic activity of the binding polypeptide. The effector moietymay be, for example but not limited to, a therapeutic or diagnosticagent. A modified binding polypeptide (e.g., an antibody) of the currentdisclosure may comprise one or more effector moieties, which may be thesame of different.

In one embodiment, the effector moiety can be of Formula (I):H₂N-Q-CON—X  Formula (I),wherein:

A) Q is NH or O; and

B) CON is a connector moiety; and

C) X is an effector moiety (e.g., a therapeutic or diagnostic agent asdefined herein).

The connector moiety connects the therapeutic agent to H₂N-Q-. Theconnector moiety can include at least one of any suitable componentsknown to those skilled in the art, including, for example, an alkylenylcomponent, a polyethylene glycol component, a poly(glycine) component, apoly(oxazoline) component, a carbonyl component, a component derivedfrom cysteinamide, a component derived from valine coupled withcitruline, and a component derived from 4-aminobenzyl carbamate, or anycombination thereof.

In another embodiment, the effector moiety of Formula (I) can be ofFormula (Ia):H₂N-Q-CH₂—C(O)—Z-X  Formula (Ia),wherein:

A) Q is NH or O; and

B) Z is -Cys-(MC)_(a)-(VC)_(b)-(PABC)_(c)-(C₁₆H₃₂O₈C₂H₄)_(f),

-   -   wherein        -   i. Cys is a component derived cysteinamide;        -   ii. MC is a component derived from maleimide;        -   iii. VC is a component derived from valine coupled with            citruline;        -   iv. PABC is a component derived from 4-aminobenzyl            carbamate;        -   v. X is an effector moiety (e.g., a therapeutic or            diagnostic agent as defined herein);        -   vi. a is 0 or 1;        -   vii. b is 0 or 1;        -   viii. c is 0 or 1; and        -   ix. f is 0 or 1

The “component derived from cysteinamide” is the point of attachment toH₂N-Q-CH₂—C(O)—. In one embodiment, the “component derived fromcysteinamide” can refer to one or more portions of the effector moietyhaving the structure:

In one embodiment, the “Cys” component of an effector moiety may includeone such portion. For example, the following structure shows an effectormoiety with one such portion (wherein the “Cys” component is indicatedwith the dotted line box):

In another embodiment, the “Cys” component of an effector moiety mayinclude two or more such portions. For example, the following moietycontains two such portions:

As can be seen from the structure, each “Cys” component bears an-(MC)_(a)-(VC)_(b)-(PABC)_(c)—(C₁₆H₃₂O₈C₂H₄)_(f)—X group.

In one embodiment, the phrase “component derived from maleimide” canrefer to any portion of the effector moiety having the structure:

wherein d is an integer from 2 to 5. The number of MC componentsincluded in any Cys-(MC)_(a)-(VC)_(b)-(PABC)_(c)-(C₁₆H₃₂O₈C₂H₄)_(f)—Xgroup in the effector moiety is indicated by subscript “a,” and can be 0or 1 In one embodiment, a is 1. In another embodiment, b is 0.

In one embodiment, the “Cys” component can be connected to the “MC”component via the sulfur atom in the “Cys” component, as indicated withthe dotted line box in the structure below:

In one embodiment, the phrase “component derived from valine coupledwith citruline” can refer to any portion of the effector moiety with thefollowing structure:

The number of VC components included in anyCys-(MC)_(a)-(VC)_(b)-(PABC)_(c)—(C₁₆H₃₂O₈C₂H₄)_(f)—X group in theeffector moiety is indicated by subscript “b,” and can be 0 or 1. In oneembodiment, b is 1. In another embodiment, b is 0.

In one embodiment, the phrase “component derived from 4-aminobenzylcarbamate” can refer to any portion of the effector moiety with thefollowing structure:

The number of PABC components included in anyCys-(MC)_(a)-(VC)_(b)-(PABC)_(c)—(C₁₆H₃₂O₈C₂H₄)_(f)—X group in theeffector moiety is indicated by subscript “c,” and can be 0 or 1. In oneembodiment, c is 1. In another embodiment, c is 0.

In one embodiment, “C₁₆H₃₂O₈C₂H₄” refers to the following structure:

The number of C₁₆H₃₂O₈ units included in anyCys-(MC)_(a)-(VC)_(b)-(PABC)_(c)—(C₁₆H₃₂O₈C₂H₄)_(f)—X group in theeffector moiety is indicated by subscript “f,” In one embodiment, fis 1. In another embodiment, f is 0.

In one embodiment, a is 1, b is 1, c is 1, and f is 0.

a) Therapeutic Effector Moieties

In certain embodiments, the binding polypeptides of the currentdisclosure are conjugated to an effector moiety comprising a therapeuticagent, e.g. a drug moiety (or prodrug thereof) or radiolabeled compound.In one embodiment the therapeutic agent is a cytotoxin. Exemplarycytotoxic therapeutic agents are set forth in Table 1 herein.

TABLE 1 Exemplary cytotoxic therapeutic agents

Further exemplary drug moieties include anti-inflammatory, anticancer,anti-infective (e.g., anti-fungal, antibacterial, anti-parasitic,anti-viral, etc.), and anesthetic therapeutic agents. In a furtherembodiment, the drug moiety is an anticancer agent. Exemplaryanti-cancer agents include, but are not limited to, cytostatics, enzymeinhibitors, gene regulators, cytotoxic nucleosides, tubulin bindingagents or tubulin inhibitors, proteasome inhibitors, hormones andhormone antagonists, anti-angiogenesis agents, and the like. Exemplarycytostatic anti-cancer agents include alkylating agents such as theanthracycline family of drugs (e.g. adriamycin, carminomycin,cyclosporin-A, chloroquine, methopterin, mithramycin, porfiromycin,streptonigrin, porfiromycin, anthracenediones, and aziridines). Othercytostatic anti-cancer agents include DNA synthesis inhibitors (e.g.,methotrexate and dichloromethotrexate, 3-amino-1,2,4-benzotriazine1,4-dioxide, aminopterin, cytosine β-D-arabinofuranoside,5-fluoro-5′-deoxyuridine, 5-fluorouracil, ganciclovir, hydroxyurea,actinomycin-D, and mitomycin C), DNA-intercalators or cross-linkers(e.g., bleomycin, carboplatin, carmustine, chlorambucil,cyclophosphamide, cis-diammineplatinum(II) dichloride (cisplatin),melphalan, mitoxantrone, and oxaliplatin), and DNA-RNA transcriptionregulators (e.g., actinomycin D, daunorubicin, doxorubicin,homoharringtonine, and idarubicin). Other exemplary cytostatic agentsthat are compatible with the present disclosure include ansamycinbenzoquinones, quinonoid derivatives (e.g. quinolones, genistein,bactacyclin), busulfan, ifosfamide, mechlorethamine, triaziquone,diaziquone, carbazilquinone, indoloquinone EO9,diaziridinyl-benzoquinone methyl DZQ, triethylenephosphoramide, andnitrosourea compounds (e.g. carmustine, lomustine, semustine).

Exemplary cytotoxic nucleoside anti-cancer agents include, but are notlimited to: adenosine arabinoside, cytarabine, cytosine arabinoside,5-fluorouracil, fludarabine, floxuridine, ftorafur, and6-mercaptopurine. Exemplary anti-cancer tubulin binding agents include,but are not limited to: taxoids (e.g. paclitaxel, docetaxel, taxane),nocodazole, rhizoxin, dolastatins (e.g. Dolastatin-10, -11, or -15),colchicine and colchicinoids (e.g. ZD6126), combretastatins (e.g.Combretastatin A-4, AVE-6032), and vinca alkaloids (e.g. vinblastine,vincristine, vindesine, and vinorelbine (navelbine)). Exemplaryanti-cancer hormones and hormone antagonists, include, but are notlimited to: corticosteroids (e.g. prednisone), progestins (e.g.hydroxyprogesterone or medroprogesterone), estrogens, (e.g.diethylstilbestrol), antiestrogens (e.g. tamoxifen), androgens (e.g.testosterone), aromatase inhibitors (e.g. aminogluthetimide),17-(allylamino)-17-demethoxygeldanamycin, 4-amino-1,8-naphthalimide,apigenin, brefeldin A, cimetidine, dichloromethylene-diphosphonic acid,leuprolide (leuprorelin), luteinizing hormone-releasing hormone,pifithrin-a, rapamycin, sex hormone-binding globulin, and thapsigargin.Exemplary anti-cancer, anti-angiogenesis, but are not limited to:compounds included Angiostatin Kl-3, DL-a-difluoromethyl-ornithine,endostatin, fumagillin, genistein, minocycline, staurosporine, and(±)-thalidomide.

Exemplary anti-cancer enzyme inhibitors include, but are not limited to,S(+)-camptothecin, curcumin, (−)-deguelin, 5,6-diCHlorobenz-imidazole1-β-D-ribofuranoside, etoposide, formestane, fostriecin, hispidin,2-imino-1-imidazolidineacetic acid (cyclocreatine), mevinolin,trichostatin A, tyrphostin AG 34, and tyrphostin AG 879.

Examplary anti-cancer gene regulators include, but are not limited to:5-aza-2′-deoxycytidine, 5-azacytidine, cholecalciferol (vitamin D3),4-hydroxytamoxifen, melatonin, mifepristone, raloxifene, trans-retinal(vitamin A aldehydes), retinoic acid, vitamin A acid, 9-cis-retinoicacid, 13-cis-retinoic acid, retinol (vitamin A), tamoxifen, andtroglitazone.

Other preferred classes of anti-cancer agents include, but are notlimited to, the pteridine family of drugs, diynenes, and thepodophyllotoxins. Particularly useful members of those classes include,for example, methopterin, podophyllotoxin, or podophyllotoxinderivatives such as etoposide or etoposide phosphate, leurosidine,vindesine, leurosine and the like.

Still other anti-cancer agents that are compatible with the teachingsherein include auristatins (e.g. auristatin E and monomethylauristan E),geldanamycin, calicheamicin, gramicidin D, maytansanoids (e.g.maytansine), neocarzinostatin, topotecan, taxanes, cytochalasin B,ethidium bromide, emetine, tenoposide, colchicin, dihydroxyanthracindione, mitoxantrone, procaine, tetracaine, lidocaine,propranolol, puromycin, and analogs or homologs thereof.

Still other anti-cancer agents that are compatible with the teachingsherein include tomaymycin derivatives, maytansine derivatives,cryptophycine derivatives, anthracycline derivatives, bisphosphonatederivatives, leptomycin derivatives, streptonigrin derivatives,auristatine derivatives, and duocarmycin derivatives

Another class of compatible anti-cancer agents that may be used as drugmoieties are radiosensitizing drugs that may be effectively directed totumor or immunoreactive cells. Such drug moeities enhance thesensitivity to ionizing radiation, thereby increasing the efficacy ofradiotherapy. Not to be limited by theory, but an antibody modified witha radiosensitizing drug moiety and internalized by the tumor cell woulddeliver the radiosensitizer nearer the nucleus where radiosensitizationwould be maximal. Antibodies which lose the radiosensitizer moiety wouldbe cleared quickly from the blood, localizing the remainingradiosensitization agent in the target tumor and providing minimaluptake in normal tissues. After clearance from the blood, adjunctradiotherapy could be administered by external beam radiation directedspecifically to the tumor, radioactivity directly implanted in thetumor, or systemic radioimmunotherapy with the same modified antibody.

In one embodiment, the therapeutic agent comprises radionuclides orradiolabels with high-energy ionizing radiation that are capable ofcausing multiple strand breaks in nuclear DNA, leading to cell death.Exemplary high-energy radionuclides include: 90Y, 125I, 131I, 123I,111In, 105Rh, 153Sm, 67Cu, 67Ga, 166Ho, 177Lu, 186Re and 188Re. Theseisotopes typically produce high energy α- or β-particles which have ashort path length. Such radionuclides kill cells to which they are inclose proximity, for example neoplastic cells to which the conjugate hasattached or has entered. They have little or no effect on non-localizedcells and are essentially non-immunogenic. Alternatively, high-energyisotopes may be generated by thermal irradiation of an otherwise stableisotope, for example as in boron neutron-capture therapy (Guan et al.,PNAS, 95: 13206-10, 1998).

In one embodiment, the therapeutic agent is selected from MMAE, MMAF,and PEGS-Do110.

Exemplary therapeutic effector moieties include the structures:

In one embodiment, the effector moiety is selected from:

In certain embodiments, the effector moiety contains more than onetherapeutic agent. These multiple therapeutic agents can be the same ordifferent.

b) Diagnostic Effector Moieties

In certain embodiments, the binding polypeptides of the currentdisclosure are conjugated to an effector moiety comprising a diagnosticagent. In one embodiment, the diagnostic agent is a detectable smallmolecule label, e.g. biotin, fluorophores, chromophores, spin resonanceprobes, or radiolabels. Exemplary fluorophores include fluorescent dyes(e.g. fluorescein, rhodamine, and the like) and other luminescentmolecules (e.g. luminal). A fluorophore may be environmentally-sensitivesuch that its fluorescence changes if it is located close to one or moreresidues in the modified binding polypeptide that undergo structuralchanges upon binding a substrate (e.g. dansyl probes). Exemplaryradiolabels include small molecules containing atoms with one or morelow sensitivity nuclei (13C, 15N, 2H, 125I, 124I, 123I, 99Tc, 43K, 52Fe,64Cu, 68Ga, 111In and the like). Preferably, the radionuclide is agamma, photon, or positron-emitting radionuclide with a half-lifesuitable to permit activity or detection after the elapsed time betweenadministration and localization to the imaging site.

In one embodiment, the diagnostic agent is a polypeptide. Exemplarydiagnostic polypeptides include enzymes with fluorogenic or chromogenicactivity, e.g. the ability to cleave a substrate which forms afluorophore or chromophore as a product (i.e. reporter proteins such asluciferase). Other diagnostic proteins may have intrinsic fluorogenic orchromogenic activity (e.g., green, red, and yellow fluorescentbioluminescent aequorin proteins from bioluminescent marine organisms)or they may comprise a protein containing one or more low-energyradioactive nuclei (13C, 15N, 2H, 125I, 124I, 123I, 99Tc, 43K, 52Fe,64Cu, 68Ga, 111In and the like).

With respect to the use of radiolabeled conjugates in conjunction withthe present disclosure, binding polypeptides of the current disclosuremay be directly labeled (such as through iodination) or may be labeledindirectly through the use of a chelating agent. As used herein, thephrases “indirect labeling” and “indirect labeling approach” both meanthat a chelating agent is covalently attached to a binding polypeptideand at least one radionuclide is associated with the chelating agent.Such chelating agents are typically referred to as bifunctionalchelating agents as they bind both the polypeptide and the radioisotope.Exemplary chelating agents comprise1-isothiocycmatobenzyl-3-methyldiothelene triaminepentaacetic acid(“MX-DTPA”) and cyclohexyl diethylenetriamine pentaacetic acid(“CHX-DTPA”) derivatives. Other chelating agents comprise P-DOTA andEDTA derivatives. Particularly preferred radionuclides for indirectlabeling include 111In and 90Y. Most imaging studies utilize 5 mCi111In-labeled antibody, because this dose is both safe and has increasedimaging efficiency compared with lower doses, with optimal imagingoccurring at three to six days after antibody administration. See, forexample, Murray, (1985), J. Nuc. Med. 26: 3328 and Carraguillo et al,(1985), J. Nuc. Med. 26: 67. A particularly preferred radionuclide fordirect labeling is 131I. Those skilled in the art will appreciate thatnon-radioactive conjugates may also be assembled depending on theselected agent to be conjugated.

In certain embodiments, the diagnostic effector moiety is a FRET(Fluorescence Resonance Energy Transfer) probe. FRET has been used for avariety of diagnostic applications including cancer diagnostics. A FRETprobe may include a cleavable linker (enzyme sensitive or pH linker)connecting the donor and acceptor moieties of the FRET probe, whereincleavage results in enhanced fluorescence (including near Infrared)(see, e.g., A. Cobos-Correa et. al. Membrane-bound FRET probe visualizesMMP12 activity in pulmonary inflammation, Nature Chemical Biology(2009), 5(9), 628-63; S. Gehrig et.al. Spatially Resolved Monitoring ofNeutrophil Elastase Activity with Ratiometric Fluorescent Reporters(2012) Angew. Chem. Int. Ed., 51, 6258-6261).

In one embodiment, the effector moiety is selected from:

c) Functionalized Effector Moieties

In certain embodiments, the effector moieties of the invention may befunctionalized to contain additional groups in addition to the effectormoiety itself. For example, the effector moiety may contain cleavablelinkers which release the effector moiety from the binding polypeptideunder particular conditions. In exemplary embodiments, the effectormoiety may include a linker that is cleavable by cellular enzymes and/oris pH sensitive. Additionally or alternatively, the effector moiety maycontain a disulfide bond that cleaved by intracellular glutathione uponuptake into the cell. Exemplary disulfide and pH senstitive linkers areprovided below:

In yet other embodiments, the effector moiety may include hydrophilicand biocompatible moieties such as poly(glycine), poly(oxazoline), orPEG moieties. Exemplary structures (“Y”) are provided below:

In certain embodiments, the effector moiety contains an aminooxy groupwhich facilitates conjugation to a binding polypeptide via a stableoxime linkage. Exemplary effector moieties containing aminooxy groupsare set forth in Table 2 herein.

TABLE 2 Exemplary aminoxy effector moieties (wherein X can be anylinker, Y is any spacer, and wherein X and/or Y are optional) Z—Y—X—Drug

W, W1 and W2 =

X =

R¹⁻⁵ = H, Alkyl or Aryl Y =

Z =

In other embodiments, the effector moiety contains a hydrazide and/orN-alkylated hydrazine group to facilitate conjugation to a bindingpolypeptide via a stable hydrazone linkage. Exemplary effector moietiescontaining aminooxy groups are set forth in Table 14 herein.

TABLE 14 Exemplary hydrazine and/or hydrazide effector moieties

d) Targeting Moieties

In certain embodiments, effector moieties comprise targeting moietiesthat specifically bind to one or more target molecules. Any type oftargeting moiety can be employed including, without limitation,proteins, nucleic acids, lipids, carbohydrates (e.g., glycans), andcombinations thereof (e.g., glycoproteins, glycopeptides, andglycolipids). In certain embodiments, the targeting moiety is acarbohydrate or glycopeptide. In certain embodiments, the targetingmoiety is a glycan. Targeting moieties can be naturally or non-naturallyoccurring molecules.

VI. Conjugation of Effector Moieties to Binding Polypeptides

In certain embodiments, effector moieties are conjugated (eitherdirectly or through a linker moiety) to an oxidized glycan (e.g., anoxidized N-linked glycan) of an altered binding polypeptide, (e.g., anengineered glycan at N114 of an antibody CH1 domain). The term “oxidizedglycan” means that an alcohol substituent on the glycan has beenoxidized, providing a carbonyl substituent. The carbonyl substituent canreact with suitable nitrogen nucleophile to form a carbon-nitrogendouble bond. For example, reaction of the carbonyl group with anaminooxy group or hydrazine group would form an oxime or hydrazine,respectively. In one embodiment, the carbonyl substituent is analdehyde. Suitable oxidized glycans include oxidized galactose andoxidized sialic acid.

In one embodiment, the modified polypeptide of Formula (II) may be ofFormula (II):Ab(Gal-C(O)H)_(x)(Gal-Sia-C(O)H)_(y)  Formula (II),wherein

-   -   A) Ab is an antibody or other binding polypeptide as defined        herein;    -   B) Gal is a component derived from galactose;    -   C) Sia is a component derived from sialic acid;    -   D) x is 0 to 5; and    -   E) y is 0 to 5,        -   wherein at least one of x and y is not 0.

Any art recognized chemistry can be employed to conjugate an effectormoiety (e.g., an effector moiety comprising a linker moiety) to a glycan(see e.g., Hermanson, G. T., Bioconjugate Techniques. Academic Press(1996), which is incorporated herein ion its entirety). In certainembodiments, a saccharide residue (e.g., a sialic acid or galactoseresidue) of the glycan is first oxidized (e.g., using sodium periodatetreatment to generate a reactive aldehyde group. This aldehyde group isreacted with effector moiety an aminooxy group or hydrazine group toform an oxime or hydrazone linker, respectively. Exemplary methodsemploying this general reaction scheme are set forth in Examples 10 to15.

In certain embodiments, the native or engineered glycans of a bindingpolypeptide are first pre-treated with a glycosyltransferase enzyme invitro to provide a terminal saccharide residue that is suitablyreactive. For example, sialylation may be achieved first using acombination of galactosyltransferase (Gal T) and sialyltransferase (SialT). In certain embodiments, biantennary glycans that lack galatose (G0For G0) or that contain only one galactose (G1F or G1) can be convertedto higher-order galactosylated or sialylated structures suitable forconjugation (G1F, G1, G2F, G2, G1S1F, G1S1, G2S1F, G2S1, G2S2F, orG2S2).

An exemplary conjugation scheme for producing sialylated glycoconjugatesis shown in FIG. 25C. Sialic acid residues are introduced enzymaticallyand site specifically into the glycan of an antibody (e.g., anengineered glycan at N114 of the CH1 domain) using a combination ofgalactosyltransferase (Gal T) and sialyltransferase (Sial T). Introducedsialic acid residues are subsequently oxidized with a low concentrationof sodium periodate to yield reactive sialic acid aldehydes suitablyreactive with drug-linkers (e.g., aminooxy drug linkers) to generateantibody drug conjugates (ADC) (e.g., oxime-linked ADCs). By controllingthe number of glycan and the number of sialic residues with in vitroremodeling, the skilled artisan may have precise control over thedrug-antibody ratio (DAR) of the ADCs. For example, if ˜1 sialic acid isadded onto a single biantennary glycan (A1F) in each of heavy chain, anantibody or binding polypeptide with a DAR of 2 can be homogeneouslyobtained.

VII. Modified Binding Polypeptides

In certain embodiments, the invention provides modified polypeptideswhich are the product of the conjugating effector moieties areconjugated (either directly or through a linker moiety) to an oxidizedglycan (e.g., an oxidized N-linked glycan) of an altered bindingpolypeptide (e.g., an engineered glycan at N114 of an antibody CH1domain).

In certain embodiments,

In one embodiment, the binding polypeptide can be of Formula (III):Ab--(Gal-C(H)═N-Q-CON—X)_(x)(Gal-Sia-C(H)═N-Q-CON—X)_(y)  Formula (III),wherein:

-   -   A) Ab is an antibody as defined herein;    -   B) Q is NH or O;    -   C) CON is a connector moiety as defined herein; and    -   D) X is a therapeutic or diagnostic agent as defined herein;    -   E) Gal is a component derived from galactose;    -   F) Sia is a component derived from sialic acid;    -   G) x is 0 to 5; and    -   H) y is 0 to 5,        -   wherein at least one of x and y is not 0.

In one embodiment, the binding polypeptide can be of Formula (III) canbe of Formula (IIIa):Ab(Gal-C(H)═N-Q-CH₂—C(O)—Z-X)_(x)(Gal-Sia-C(H)═N-Q-CH₂—C(O)—Z-X)_(y)  Formula(IIIa),wherein:

-   -   A) Ab is an antibody;    -   B) Q is NH or O;    -   C) Z is Cys-(MC)_(a)-(VC)_(b)-(PABC)_(c)—(C₁₆H₃₂O₈C₂H₄)_(f)—,        wherein        -   i. Cys is a component derived cysteinamide;        -   ii. MC is a component derived from maleimide;        -   iii. VC is a component derived from valine coupled with            citruline;        -   iv. PABC is a component derived from 4-aminobenzyl            carbamate;        -   v. X is an effector moiety (e.g., a therapeutic or            diagnostic agent as defined herein);        -   vi. a is 0 or 1;        -   vii. b is 0 or 1;        -   viii. c is 0 or 1; and        -   ix. f is 0 or 1;    -   D) X is a therapeutic agent as defined herein;    -   E) Gal is a component derived from galactose;    -   F) Sia is a component derived from sialic acid;    -   G) x is 0 to 5; and    -   H) y is 0 to 5,        -   wherein at least one of x and y is not 0.

It is to be understood that the Formula (III) is not intended to implythat the antibody, the Gal substituent, and the Gal-Sia subsistent areconnected in a chain-like manner. Rather, when such substituents arepresent, the antibody is connected directly to each substituent. Forexample, a binding polypeptide of Formula (III) in which x is 1 and y is2 could have the arrangement shown below:

The CON substituent in Formula (III) and components therein are asdescribed with regard to Formula (I) for effector moieties.

In one embodiment, Q is NH. In another embodiment, Q is O.

In one embodiment, x is O.

The antibody Ab of Formula (III) may be any suitable antibody asdescribed herein.

In one embodiment, there is provided a method for preparing the bindingpolypeptide of Formula (III), the method comprising reacting an effectormoiety of Formula (I):NH₂-Q-CON—X  Formula (I),wherein:

-   -   A) Q is NH or O;    -   B) CON is a connector moiety; and    -   C) X is an effector moiety (e.g., a therapeutic or diagnostic        agent as defined herein),        with a modified antibody of Formula (II)        Ab(OXG)_(r)  Formula (II)        wherein    -   A) OXG is an oxidized glycan; and    -   B) r is selected from 0 to 4;

In one embodiment, there is provided a method for preparing the bindingpolypeptide of Formula (III), the method comprising reacting an effectormoiety of Formula (I):NH₂-Q-CON—X  Formula (I),wherein:

-   -   A) Q is NH or O;    -   B) CON is a connector moiety; and    -   C) X is an effector moiety (e.g., a therapeutic or diagnostic        agent as defined herein),        with a modified antibody of Formula (IIa)        Ab(Gal-C(O)H)_(x)(Gal-Sia-C(O)H)_(y)  Formula (IIa),        wherein    -   A) Ab is an antibody as described herein;    -   B) Gal is a component derived from galactose;    -   C) Sia is a component derived from sialic acid;    -   D) x is 0 to 5; and    -   E) y is 0 to 5,

wherein at least one of x and y is not 0.

In certain embodiments, effector moieties are conjugated (eitherdirectly or through a linker moiety) to an oxidized glycan (e.g., anoxidized N-linked glycan) of an altered binding polypeptide, (e.g., anengineered glycan at N114 of an antibody CH1 domain). The term “oxidizedglycan” means that an alcohol substituent on the glycan has beenoxidized, providing a carbonyl substituent. The carbonyl substituent canreact with suitable nitrogen nucleophile to form a carbon-nitrogendouble bond. For example, reaction of the carbonyl group with anaminooxy group or hydrazine group would form an oxime or hydrazine,respectively. In one embodiment, the carbonyl substituent is analdehyde. Suitable oxidized glycans include oxidized galactose andoxidized sialic acid.

In one embodiment, the modified polypeptide of Formula (II) may be ofFormula (II):Ab(Gal-C(O)H)_(x)(Gal-Sia-C(O)H)_(y)  Formula (II),wherein

-   -   A) Ab is an antibody or other binding polypeptide as defined        herein;    -   B) Gal is a component derived from galactose;    -   C) Sia is a component derived from sialic acid;    -   D) x is 0 to 5; and    -   E) y is 0 to 5,        -   wherein at least one of x and y is not 0.

Any art recognized chemistry can be employed to conjugate an effectormoiety (e.g., an effector moiety comprising a linker moiety) to a glycan(see e.g., Hermanson, G. T., Bioconjugate Techniques. Academic Press(1996), which is incorporated herein in its entirety). In certainembodiments, a saccharide residue (e.g., a sialic acid or galactoseresidue) of the glycan is first oxidized (e.g., using sodium periodatetreatment) to generate a reactive aldehyde group. This aldehyde group isreacted with an aminooxy group or hydrazine group of effector moiety toform an oxime or hydrazone linker, respectively. Exemplary methodsemploying this general reaction scheme are set forth in Examples 10 to15.

In certain embodiments, the native or engineered glycans of a bindingpolypeptide are first pre-treated with a glycosyltransferase enzyme invitro to provide a terminal saccharide residue that is suitablyreactive. For example, sialylation may be achieved first using acombination of galactosyltransferase (Gal T) and sialyltransferase (SialT). In certain embodiments, biantennary glycans that lack galatose (G0For G0) or that contain only one galactose (G1F or G1) can be convertedto higher-order galactosylated or sialylated structures suitable forconjugation (G1F, G1, G2F, G2, G1S1F, G1S1, G2S1F, G2S1, G2S2F, orG2S2).

An exemplary conjugation scheme for producing sialylated glycoconjugatesis shown in FIG. 25C. Sialic acid residues are introduced enzymaticallyand site specifically into the glycan of an antibody (e.g., anengineered glycan at N114 of the CH1 domain) using a combination ofgalactosyltransferase (Gal T) and sialyltransferase (Sial T). Introducedsialic acid residues are subsequently oxidized with a low concentrationof sodium periodate to yield reactive sialic acid aldehydes suitablyreactive with drug-linkers (e.g., aminooxy drug linkers) to generateantibody drug conjugates (ADC) (e.g., oxime-linked ADCs). By controllingthe number of glycan and the number of sialic residues with in vitroremodeling, the skilled artisan may have precise control over thedrug-antibody ratio (DAR) of the ADCs. For example, if ˜1 sialic acid isadded onto a single biantennary glycan (A1F) in each of heavy chain, anantibody or binding polypeptide with a DAR of 2 can be homogeneouslyobtained.

VII. Modified Binding Polypeptides

In certain embodiments, the invention provides modified polypeptideswhich are the product of the conjugating effector moieties areconjugated (either directly or through a linker moiety) to an oxidizedglycan (e.g., an oxidized N-linked glycan) of an altered bindingpolypeptide (e.g., an engineered glycan at N114 of an antibody CH1domain).

In certain embodiments,

In one embodiment, the binding polypeptide can be of Formula (III):Ab--(Gal-C(H)═N-Q-CON—X)_(x)(Gal-Sia-C(H)═N-Q-CON—X)_(y)  Formula (III),wherein:

-   -   A) Ab is an antibody as defined herein;    -   B) Q is NH or O;    -   C) CON is a connector moiety as defined herein; and    -   D) X is an effector moiety (e.g., a therapeutic or diagnostic        agent as defined herein);    -   E) Gal is a component derived from galactose;    -   F) Sia is a component derived from sialic acid;    -   G) x is 0 to 5; and    -   H) y is 0 to 5,        -   wherein at least one of x and y is not 0.

In one embodiment, the binding polypeptide can be of Formula (III) canbe of Formula (IIIa):Ab(Gal-C(H)═N-Q-CH₂—C(O)—Z-X)_(x)(Gal-Sia-C(H)═N-Q-CH₂—C(O)—Z-X)_(y)  Formula(IIIa),wherein:

-   -   A) Ab is an antibody;    -   B) Q is NH or O;    -   C) Z is Cys-(MC)_(a)—(VC)_(b)—(PABC)_(c)—(C₁₆H₃₂O₈C₂H₄)_(f)—,        wherein        -   i. Cys is a component derived cysteinamide;        -   ii. MC is a component derived from maleimide;        -   iii. VC is a component derived from valine coupled with            citruline;        -   iv. PABC is a component derived from 4-aminobenzyl            carbamate;        -   v. X is a therapeutic or diagnostic agent as defined herein;        -   vi. a is 0 or 1;        -   vii. b is 0 or 1;        -   viii. c is 0 or 1; and        -   ix. f is 0 or 1;    -   D) X is a therapeutic agent as defined herein;    -   E) Gal is a component derived from galactose;    -   F) Sia is a component derived from sialic acid;    -   G) x is 0 to 5; and    -   H) y is 0 to 5,        -   wherein at least one of x and y is not 0.

It is to be understood that the Formula (III) is not intended to implythat the antibody, the Gal substituent, and the Gal-Sia subsituent areconnected in a chain-like manner. Rather, when such substituents arepresent, the antibody is connected directly to each substituent. Forexample, a binding polypeptide of Formula (III) in which x is 1 and y is2 could have the arrangement shown below:

The CON substituent in Formula (III) and components therein are asdescribed with regard to Formula (I) for effector moieties.

In one embodiment, Q is NH. In another embodiment, Q is O.

In one embodiment, x is 0.

The antibody Ab of Formula (III) may be any suitable antibody asdescribed herein.

In one embodiment, there is provided a method for preparing the bindingpolypeptide of Formula (III), the method comprising reacting an effectormoiety of Formula (I):NH₂-Q-CON—X  Formula (I),wherein:

A) Q is NH or O;

B) CON is a connector moiety; and

C) X is a therapeutic or diagnostic agent as defined herein,

with a modified antibody of Formula (II)Ab(OXG)_(r)  Formula (II)wherein

-   -   A) OXG is an oxidized glycan; and    -   B) r is selected from 0 to 4;

In one embodiment, there is provided a method for preparing the bindingpolypeptide of Formula (III), the method comprising reacting an effectormoiety of Formula (I):NH₂-Q-CON—X  Formula (I),wherein:

-   -   A) Q is NH or O;    -   B) CON is a connector moiety; and    -   C) X is an effector moiety (e.g., a therapeutic or diagnostic        agent as defined herein),        with a modified antibody of Formula (IIa)        Ab(Gal-C(O)H)_(x)(Gal-Sia-C(O)H)_(y)  Formula (IIa),        wherein    -   A) Ab is an antibody as described herein;    -   B) Gal is a component derived from galactose;    -   C) Sia is a component derived from sialic acid;    -   D) x is 0 to 5; and    -   E) y is 0 to 5,        -   wherein at least one of x and y is not 0.            VIII. Methods of Treatment with Modified Antibodies

In one aspect, the invention provides methods of treating or diagnosinga patient in thereof comprising administering an effective amount abinding polypeptide disclosed herein. Preferred embodiments of thepresent disclosure provide kits and methods for the diagnosis and/ortreatment of disorders, e.g., neoplastic disorders in a mammaliansubject in need of such treatment. Preferably, the subject is a human.

The binding polypeptides of the current disclosure are useful in anumber of different applications. For example, in one embodiment, thesubject binding polypeptide s are useful for reducing or eliminatingcells bearing an epitope recognized by the binding domain of the bindingpolypeptide. In another embodiment, the subject binding polypeptides areeffective in reducing the concentration of or eliminating solubleantigen in the circulation. In one embodiment, the binding polypeptidesmay reduce tumor size, inhibit tumor growth and/or prolong the survivaltime of tumor-bearing animals. Accordingly, this disclosure also relatesto a method of treating tumors in a human or other animal byadministering to such human or animal an effective, non-toxic amount ofmodified antibody. One skilled in the art would be able, by routineexperimentation, to determine what an effective, non-toxic amount ofmodified binding polypeptide would be for the purpose of treatingmalignancies. For example, a therapeutically active amount of a modifiedantibody or fragments thereof may vary according to factors such as thedisease stage (e.g., stage I versus stage IV), age, sex, medicalcomplications (e.g., immunosuppressed conditions or diseases) and weightof the subject, and the ability of the modified antibody to elicit adesired response in the subject. The dosage regimen may be adjusted toprovide the optimum therapeutic response. For example, several divideddoses may be administered daily, or the dose may be proportionallyreduced as indicated by the exigencies of the therapeutic situation.

In general, the compositions provided in the current disclosure may beused to prophylactically or therapeutically treat any neoplasmcomprising an antigenic marker that allows for the targeting of thecancerous cells by the modified antibody.

IX. Methods of Administering Modified Antibodies or Fragments Thereof

Methods of preparing and administering binding polypeptides of thecurrent disclosure to a subject are well known to or are readilydetermined by those skilled in the art. The route of administration ofthe binding polypeptides of the current disclosure may be oral,parenteral, by inhalation or topical. The term parenteral as used hereinincludes intravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, rectal or vaginal administration. The intravenous,intraarterial, subcutaneous and intramuscular forms of parenteraladministration are generally preferred. While all these forms ofadministration are clearly contemplated as being within the scope of thecurrent disclosure, a form for administration would be a solution forinjection, in particular for intravenous or intraarterial injection ordrip. Usually, a suitable pharmaceutical composition for injection maycomprise a buffer (e.g. acetate, phosphate or citrate buffer), asurfactant (e.g. polysorbate), optionally a stabilizer agent (e.g. humanalbumin), etc. However, in other methods compatible with the teachingsherein, the modified antibodies can be delivered directly to the site ofthe adverse cellular population thereby increasing the exposure of thediseased tissue to the therapeutic agent.

In one embodiment, the binding polypeptide that is administered is abinding polypeptide of Formula (III):Ab(Gal-C(H)═N-Q-CON—X)_(x)(Gal-Sia-C(H)═N-Q-CON—X)_(y)  Formula (III),wherein:

-   -   A) Ab is an antibody as defined herein;    -   B) Q is NH or O;    -   C) CON is a connector moiety as defined herein; and    -   D) X is an effector moiety (e.g., a therapeutic or diagnostic        agent as defined herein);    -   E) Gal is a component derived from galactose;    -   F) Sia is a component derived from sialic acid;    -   G) x is 0 to 5; and    -   H) y is 0 to 5,        -   wherein at least one of x and y is not 0.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. In the compositions and methods of the current disclosure,pharmaceutically acceptable carriers include, but are not limited to,0.01-0.1 M and preferably 0.05M phosphate buffer or 0.8% saline. Othercommon parenteral vehicles include sodium phosphate solutions, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers, such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present such asfor example, antimicrobials, antioxidants, chelating agents, and inertgases and the like. More particularly, pharmaceutical compositionssuitable for injectable use include sterile aqueous solutions (wherewater soluble) or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersions. In suchcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and will preferably be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants.

Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

In any case, sterile injectable solutions can be prepared byincorporating an active compound (e.g., a modified binding polypeptideby itself or in combination with other active agents) in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated herein, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle, which contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying, which yields a powder of an activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof. The preparations for injections areprocessed, filled into containers such as ampoules, bags, bottles,syringes or vials, and sealed under aseptic conditions according tomethods known in the art. Further, the preparations may be packaged andsold in the form of a kit such as those described in co-pending U.S.Ser. No. 09/259,337 and U.S. Ser. No. 09/259,338 each of which isincorporated herein by reference. Such articles of manufacture willpreferably have labels or package inserts indicating that the associatedcompositions are useful for treating a subject suffering from, orpredisposed to autoimmune or neoplastic disorders.

Effective doses of the compositions of the present disclosure, for thetreatment of the above described conditions vary depending upon manydifferent factors, including means of administration, target site,physiological state of the patient, whether the patient is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic. Usually, the patient is a human butnon-human mammals including transgenic mammals can also be treated.Treatment dosages may be titrated using routine methods known to thoseof skill in the art to optimize safety and efficacy.

For passive immunization with a binding polypeptide, the dosage canrange, e.g., from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2mg/kg, etc.), of the host body weight. For example dosages can be 1mg/kg body weight or 10 mg/kg body weight or within the range of 1-10mg/kg, preferably at least 1 mg/kg. Doses intermediate in the aboveranges are also intended to be within the scope of the currentdisclosure. Subjects can be administered such doses daily, onalternative days, weekly or according to any other schedule determinedby empirical analysis. An exemplary treatment entails administration inmultiple dosages over a prolonged period, for example, of at least sixmonths. Additional exemplary treatment regimens entail administrationonce per every two weeks or once a month or once every 3 to 6 months.Exemplary dosage schedules include 1-10 mg/kg or 15 mg/kg on consecutivedays, 30 mg/kg on alternate days or 60 mg/kg weekly. In some methods,two or more monoclonal antibodies with different binding specificitiesare administered simultaneously, in which case the dosage of eachantibody administered falls within the ranges indicated.

Binding polypeptides of the current disclosure can be administered onmultiple occasions. Intervals between single dosages can be weekly,monthly or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of modified binding polypeptide or antigen in thepatient. In some methods, dosage is adjusted to achieve a plasmamodified binding polypeptide concentration of 1-1000 μg/ml and in somemethods 25-300 μg/ml. Alternatively, binding polypeptides can beadministered as a sustained release formulation, in which case lessfrequent administration is required. For antibodies, dosage andfrequency vary depending on the half-life of the antibody in thepatient. In general, humanized antibodies show the longest half-life,followed by chimeric antibodies and nonhuman antibodies.

The dosage and frequency of administration can vary depending on whetherthe treatment is prophylactic or therapeutic. In prophylacticapplications, compositions containing the present antibodies or acocktail thereof are administered to a patient not already in thedisease state to enhance the patient's resistance. Such an amount isdefined to be a “prophylactic effective dose.” In this use, the preciseamounts again depend upon the patient's state of health and generalimmunity, but generally range from 0.1 to 25 mg per dose, especially 0.5to 2.5 mg per dose. A relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage (e.g., from about 1to 400 mg/kg of antibody per dose, with dosages of from 5 to 25 mg beingmore commonly used for radioimmunoconjugates and higher doses forcytotoxin-drug modified antibodies) at relatively short intervals issometimes required until progression of the disease is reduced orterminated, and preferably until the patient shows partial or completeamelioration of disease symptoms. Thereafter, the patient can beadministered a prophylactic regime.

Binding polypeptides of the current disclosure can optionally beadministered in combination with other agents that are effective intreating the disorder or condition in need of treatment (e.g.,prophylactic or therapeutic). Effective single treatment dosages (i.e.,therapeutically effective amounts) of 90Y-labeled modified antibodies ofthe current disclosure range from between about 5 and about 75 mCi, morepreferably between about 10 and about 40 mCi. Effective single treatmentnon-marrow ablative dosages of 131I-modified antibodies range frombetween about 5 and about 70 mCi, more preferably between about 5 andabout 40 mCi. Effective single treatment ablative dosages (i.e., mayrequire autologous bone marrow transplantation) of 131I-labeledantibodies range from between about 30 and about 600 mCi, morepreferably between about 50 and less than about 500 mCi. In conjunctionwith a chimeric antibody, owing to the longer circulating half-lifevis-a-vis murine antibodies, an effective single treatment non-marrowablative dosages of iodine-131 labeled chimeric antibodies range frombetween about 5 and about 40 mCi, more preferably less than about 30mCi. Imaging criteria for, e.g., the 111In label, are typically lessthan about 5 mCi.

While the binding polypeptides may be administered as describedimmediately above, it must be emphasized that in other embodimentsbinding may be administered to otherwise healthy patients as a firstline therapy. In such embodiments the binding polypeptides may beadministered to patients having normal or average red marrow reservesand/or to patients that have not, and are not, undergoing. As usedherein, the administration of modified antibodies or fragments thereofin conjunction or combination with an adjunct therapy means thesequential, simultaneous, coextensive, concurrent, concomitant orcontemporaneous administration or application of the therapy and thedisclosed antibodies. Those skilled in the art will appreciate that theadministration or application of the various components of the combinedtherapeutic regimen may be timed to enhance the overall effectiveness ofthe treatment. For example, chemotherapeutic agents could beadministered in standard, well known courses of treatment followedwithin a few weeks by radioimmunoconjugates of the present disclosure.Conversely, cytotoxin associated binding polypeptides could beadministered intravenously followed by tumor localized external beamradiation. In yet other embodiments, the modified binding polypeptidemay be administered concurrently with one or more selectedchemotherapeutic agents in a single office visit. A skilled artisan(e.g. an experienced oncologist) would be readily be able to discerneffective combined therapeutic regimens without undue experimentationbased on the selected adjunct therapy and the teachings of the instantspecification.

In this regard it will be appreciated that the combination of thebinding polypeptides and the chemotherapeutic agent may be administeredin any order and within any time frame that provides a therapeuticbenefit to the patient. That is, the chemotherapeutic agent and bindingpolypeptides may be administered in any order or concurrently. Inselected embodiments the binding polypeptides of the present disclosurewill be administered to patients that have previously undergonechemotherapy. In yet other embodiments, the binding polypeptides and thechemotherapeutic treatment will be administered substantiallysimultaneously or concurrently. For example, the patient may be giventhe binding polypeptides while undergoing a course of chemotherapy. Inpreferred embodiments the modified antibody will be administered withinone year of any chemotherapeutic agent or treatment. In other preferredembodiments the binding polypeptides will be administered within 10, 8,6, 4, or 2 months of any chemotherapeutic agent or treatment. In stillother preferred embodiments the binding polypeptide will be administeredwithin 4, 3, 2, or 1 week(s) of any chemotherapeutic agent or treatment.In yet other embodiments the binding polypeptides will be administeredwithin 5, 4, 3, 2, or 1 day(s) of the selected chemotherapeutic agent ortreatment. It will further be appreciated that the two agents ortreatments may be administered to the patient within a matter of hoursor minutes (i.e. substantially simultaneously).

It will further be appreciated that the binding polypeptides of thecurrent disclosure may be used in conjunction or combination with anychemotherapeutic agent or agents (e.g. to provide a combined therapeuticregimen) that eliminates, reduces, inhibits or controls the growth ofneoplastic cells in vivo. Exemplary chemotherapeutic agents that arecompatible with the current disclosure include alkylating agents, vincaalkaloids (e.g., vincristine and vinblastine), procarbazine,methotrexate and prednisone. The four-drug combination MOPP(mechlethamine (nitrogen mustard), vincristine (Oncovin), procarbazineand prednisone) is very effective in treating various types of lymphomaand comprises a preferred embodiment of the present disclosure. InMOPP-resistant patients, ABVD (e.g., adriamycin, bleomycin, vinblastineand dacarbazine), ChIVPP (CH1orambucil, vinblastine, procarbazine andprednisone), CABS (lomustine, doxorubicin, bleomycin andstreptozotocin), MOPP plus ABVD, MOPP plus ABV (doxorubicin, bleomycinand vinblastine) or BCVPP (carmustine, cyclophosphamide, vinblastine,procarbazine and prednisone) combinations can be used. Arnold S.Freedman and Lee M. Nadler, Malignant Lymphomas, in HARRISON'SPRINCIPLES OF INTERNAL MEDICINE 1774-1788 (Kurt J. Isselbacher et al,eds., 13th ed. 1994) and V. T. DeVita et al, (1997) and the referencescited therein for standard dosing and scheduling. These therapies can beused unchanged, or altered as needed for a particular patient, incombination with one or more binding polypeptides of the currentdisclosure as described herein.

Additional regimens that are useful in the context of the presentdisclosure include use of single alkylating agents such ascyclophosphamide or chlorambucil, or combinations such as CVP(cyclophosphamide, vincristine and prednisone), CHOP (CVP anddoxorubicin), C-MOPP (cyclophosphamide, vincristine, prednisone andprocarbazine), CAP-BOP (CHOP plus procarbazine and bleomycin), m-BACOD(CHOP plus methotrexate, bleomycin and leucovorin), ProMACE-MOPP(prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide andleucovorin plus standard MOPP), ProMACE-CytaBOM (prednisone,doxorubicin, cyclophosphamide, etoposide, cytarabine, bleomycin,vincristine, methotrexate and leucovorin) and MACOP-B (methotrexate,doxorubicin, cyclophosphamide, vincristine, fixed dose prednisone,bleomycin and leucovorin). Those skilled in the art will readily be ableto determine standard dosages and scheduling for each of these regimens.CHOP has also been combined with bleomycin, methotrexate, procarbazine,nitrogen mustard, cytosine arabinoside and etoposide. Other compatiblechemotherapeutic agents include, but are not limited to,2-Chlorodeoxyadenosine (2-CDA), 2′-deoxycoformycin and fludarabine.

For patients with intermediate- and high-grade NHL, who fail to achieveremission or relapse, salvage therapy is used. Salvage therapies employdrugs such as cytosine arabinoside, carboplatin, cisplatin, etoposideand ifosfamide given alone or in combination. In relapsed or aggressiveforms of certain neoplastic disorders the following protocols are oftenused: IMVP-16 (ifosfamide, methotrexate and etoposide), MIME(methyl-gag, ifosfamide, methotrexate and etoposide), DHAP(dexamethasone, high dose cytarabine and cisplatin), ESHAP (etoposide,methylpredisolone, HD cytarabine, cisplatin), CEPP(B) (cyclophosphamide,etoposide, procarbazine, prednisone and bleomycin) and CAMP (lomustine,mitoxantrone, cytarabine and prednisone) each with well-known dosingrates and schedules.

The amount of chemotherapeutic agent to be used in combination with themodified antibodies of the current disclosure may vary by subject or maybe administered according to what is known in the art. See for example,Bruce A Chabner et al, Antineoplastic Agents, in GOODMAN & GILMAN'S THEPHARMACOLOGICAL BASIS OF THERAPEUTICS 1233-1287 ((Joel G. Hardman et al,eds., 9th ed. 1996).

As previously discussed, the binding polypeptides of the presentdisclosure, immunoreactive fragments or recombinants thereof may beadministered in a pharmaceutically effective amount for the in vivotreatment of mammalian disorders. In this regard, it will be appreciatedthat the disclosed binding polypeptides will be formulated to facilitateadministration and promote stability of the active agent.

Preferably, pharmaceutical compositions in accordance with the presentdisclosure comprise a pharmaceutically acceptable, non-toxic, sterilecarrier such as physiological saline, nontoxic buffers, preservativesand the like. For the purposes of the instant application, apharmaceutically effective amount of the modified binding polypeptide,immunoreactive fragment or recombinant thereof, conjugated orunconjugated to a therapeutic agent, shall be held to mean an amountsufficient to achieve effective binding to an antigen and to achieve abenefit, e.g., to ameliorate symptoms of a disease or disorder or todetect a substance or a cell. In the case of tumor cells, the modifiedbinding polypeptide will be preferably be capable of interacting withselected immunoreactive antigens on neoplastic or immunoreactive cellsand provide for an increase in the death of those cells. Of course, thepharmaceutical compositions of the present disclosure may beadministered in single or multiple doses to provide for apharmaceutically effective amount of the modified binding polypeptide.

In keeping with the scope of the present disclosure, the bindingpolypeptides of the disclosure may be administered to a human or otheranimal in accordance with the aforementioned methods of treatment in anamount sufficient to produce a therapeutic or prophylactic effect. Thebinding polypeptides of the disclosure can be administered to such humanor other animal in a conventional dosage form prepared by combining theantibody of the disclosure with a conventional pharmaceuticallyacceptable carrier or diluent according to known techniques. It will berecognized by one of skill in the art that the form and character of thepharmaceutically acceptable carrier or diluent is dictated by the amountof active ingredient with which it is to be combined, the route ofadministration and other well-known variables. Those skilled in the artwill further appreciate that a cocktail comprising one or more speciesof binding polypeptides described in the current disclosure may prove tobe particularly effective.

X. Expression of Binding Polypeptides

In one aspect, the invention provides polynucleotides encoding thebinding polypeptides disclosed herein. A method of making a bindingpolypeptide comprising expressing these polynucleotides are alsoprovided.

Polynucleotides encoding the binding polypeptides disclosed herein aretypically inserted in an expression vector for introduction into hostcells that may be used to produce the desired quantity of the claimedantibodies, or fragments thereof. Accordingly, in certain aspects, theinvention provides expression vectors comprising polynucleotidesdisclosed herein and host cells comprising these vectors andpolynucleotides.

The term “vector” or “expression vector” is used herein for the purposesof the specification and claims, to mean vectors used in accordance withthe present invention as a vehicle for introducing into and expressing adesired gene in a cell. As known to those skilled in the art, suchvectors may easily be selected from the group consisting of plasmids,phages, viruses and retroviruses. In general, vectors compatible withthe instant invention will comprise a selection marker, appropriaterestriction sites to facilitate cloning of the desired gene and theability to enter and/or replicate in eukaryotic or prokaryotic cells.

Numerous expression vector systems may be employed for the purposes ofthis invention. For example, one class of vector utilizes DNA elementswhich are derived from animal viruses such as bovine papilloma virus,polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses(RSV, MMTV or MOMLV), or SV40 virus. Others involve the use ofpolycistronic systems with internal ribosome binding sites.Additionally, cells which have integrated the DNA into their chromosomesmay be selected by introducing one or more markers which allow selectionof transfected host cells. The marker may provide for prototrophy to anauxotrophic host, biocide resistance (e.g., antibiotics) or resistanceto heavy metals such as copper. The selectable marker gene can either bedirectly linked to the DNA sequences to be expressed, or introduced intothe same cell by cotransformation. Additional elements may also beneeded for optimal synthesis of mRNA. These elements may include signalsequences, splice signals, as well as transcriptional promoters,enhancers, and termination signals. In particularly preferredembodiments the cloned variable region genes are inserted into anexpression vector along with the heavy and light chain constant regiongenes (preferably human) synthetic as discussed above.

In other preferred embodiments the binding polypeptides of the inventionmay be expressed using polycistronic constructs. In such expressionsystems, multiple gene products of interest such as heavy and lightchains of antibodies may be produced from a single polycistronicconstruct. These systems advantageously use an internal ribosome entrysite (IRES) to provide relatively high levels of polypeptides of theinvention in eukaryotic host cells. Compatible IRES sequences aredisclosed in U.S. Pat. No. 6,193,980 which is incorporated by referenceherein. Those skilled in the art will appreciate that such expressionsystems may be used to effectively produce the full range ofpolypeptides disclosed in the instant application.

More generally, once a vector or DNA sequence encoding an antibody, orfragment thereof, has been prepared, the expression vector may beintroduced into an appropriate host cell. That is, the host cells may betransformed. Introduction of the plasmid into the host cell can beaccomplished by various techniques well known to those of skill in theart. These include, but are not limited to, transfection (includingelectrophoresis and electroporation), protoplast fusion, calciumphosphate precipitation, cell fusion with enveloped DNA, microinjection,and infection with intact virus. See, Ridgway, A. A. G. “MammalianExpression Vectors” Chapter 24.2, pp. 470-472 Vectors, Rodriguez andDenhardt, Eds. (Butterworths, Boston, Mass. 1988). Most preferably,plasmid introduction into the host is via electroporation. Thetransformed cells are grown under conditions appropriate to theproduction of the light chains and heavy chains, and assayed for heavyand/or light chain protein synthesis. Exemplary assay techniques includeenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), orflourescence-activated cell sorter analysis (FACS), immunohistochemistryand the like.

As used herein, the term “transformation” shall be used in a broad senseto refer to the introduction of DNA into a recipient host cell thatchanges the genotype and consequently results in a change in therecipient cell.

Along those same lines, “host cells” refers to cells that have beentransformed with vectors constructed using recombinant DNA techniquesand encoding at least one heterologous gene. In descriptions ofprocesses for isolation of polypeptides from recombinant hosts, theterms “cell” and “cell culture” are used interchangeably to denote thesource of antibody unless it is clearly specified otherwise. In otherwords, recovery of polypeptide from the “cells” may mean either fromspun down whole cells, or from the cell culture containing both themedium and the suspended cells.

In one embodiment, the host cell line used for antibody expression is ofmammalian origin; those skilled in the art can determine particular hostcell lines which are best suited for the desired gene product to beexpressed therein. Exemplary host cell lines include, but are notlimited to, DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus),HELA (human cervical carcinoma), CVI (monkey kidney line), COS (aderivative of CVI with SV40 T antigen), R1610 (Chinese hamsterfibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line),SP2/O (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI(human lymphocyte), 293 (human kidney). In one embodiment, the cell lineprovides for altered glycosylation, e.g., afucosylation, of theantibodyexpressed therefrom (e.g., PER.C6® (Crucell) or FUT8-knock-outCHO cell lines (Potelligent® Cells) (Biowa, Princeton, N.J.)). In oneembodiment NS0 cells may be used. CHO cells are particularly preferred.Host cell lines are typically available from commercial services, theAmerican Tissue Culture Collection or from published literature.

In vitro production allows scale-up to give large amounts of the desiredpolypeptides. Techniques for mammalian cell cultivation under tissueculture conditions are known in the art and include homogeneoussuspension culture, e.g. in an airlift reactor or in a continuousstirrer reactor, or immobilized or entrapped cell culture, e.g. inhollow fibers, microcapsules, on agarose microbeads or ceramiccartridges. If necessary and/or desired, the solutions of polypeptidescan be purified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose and/or (immuno-) affinity chromatography.

Genes encoding the binding polypeptides of the invention can also beexpressed non-mammalian cells such as bacteria or yeast or plant cells.In this regard it will be appreciated that various unicellularnon-mammalian microorganisms such as bacteria can also be transformed;i.e. those capable of being grown in cultures or fermentation. Bacteria,which are susceptible to transformation, include members of theenterobacteriaceae, such as strains of Escherichia coli or Salmonella;Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, andHaemophilus influenzae. It will further be appreciated that, whenexpressed in bacteria, the polypeptides can become part of inclusionbodies. The polypeptides must be isolated, purified and then assembledinto functional molecules.

In addition to prokaryotes, eukaryotic microbes may also be used.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among eukaryotic microorganisms although a number of other strainsare commonly available. For expression in Saccharomyces, the plasmidYRp7, for example, (Stinchcomb et al., Nature, 282:39 (1979); Kingsmanet al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)) iscommonly used. This plasmid already contains the TRP1 gene whichprovides a selection marker for a mutant strain of yeast lacking theability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1(Jones, Genetics, 85:12 (1977)). The presence of the trpl lesion as acharacteristic of the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan.

EXAMPLES

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting. The contents ofSequence Listing, figures and all references, patents, and publishedpatent applications cited throughout this application are expresslyincorporated herein by reference.

Example 1 Design, Preparation, and Characterization of 2C3 Anti-CD-52Hyperglycosylation Antibody Mutants

Multiple hyperglycosylation mutations were designed in the heavy chainof the anti-CD-52 antibody, 2C3, for the purpose of adding a bulky groupto an interaction interface (e.g., the FcRn binding site to modulateantibody pharmacokinetics), for modulating antibody effector function bychanging its interaction with FcγRs, or to introduce a novelcross-linking site subsequence chemical modification for effector moietyconjugation, including but not limited to, drugs, toxins, cytotoxicagents, and radionucleotides. The hyperglycosylated 2C3 mutants are setforth in Table 3.

TABLE 3 Hyperglycosylated 2C3 anti-CD-52 mutants Mutation DesiredBenefit Applications A114N Glycosylation at Asn-Ser- 1) Control Thr 2)Effector moiety conjugation Y436T Glycosylation at Asn434 1) Transplantand other Inhibition of FcRn binding indications which need shorthalf-life Y436S Glycosylation at Asn434 1) Transplant and otherInhibition of FcRn binding indications which need short half-life S440NGlycosylation at Asn-Leu- 1) Control Ser 2) Effector moiety conjugationS442N Glycosylation at Asn-Leu- 1) Control Ser 2) Effector moietyconjugation Add NGT Glycosylation 1) Control to C- 2) Effector moietyconjugation terminal S298N/ Glycosylation at Asn298 1) Reduce effectorfunction Y300S Reduced effector function 2) Effector moiety conjugation1A. Creation of 2C3 Anti-CD-52 Antibody Hyperglycosylation Mutants

The A114N mutation, designated based upon the Kabat numbering system,was introduced into the CH1 domain of 2C3 by mutagenic PCR. To createthe full-length antibody, the VH domain plus the mutated A114N residuewas inserted by ligation independent cloning (LIC) into thepENTR-LIC-IgG1 vector encoding antibody CH domains 1-3. All othermutations were introduced on pENTR-LIC-IgG1 by site-directed mutagenesiswith a QuikChange site-directed mutagenesis kit (Agilent Technologies,Inc., Santa Clara, Calif., USA). The WT 2C3 VH was cloned into mutatedvectors by LIC. Full-length mutants were cloned into the pCEP4(−E+I)Destexpression vector by Gateway cloning. Fc mutations were designated basedon the EU numbering system. Mutations were confirmed by DNA sequencingAmino acid sequences of the WT 2C3 heavy and light chains and themutated 2C3 heavy chains are set forth in Table 4. Mutated amino acidsare highlighted in gray and the consensus glycosylation target sitescreated by the mutation are underlined.

TABLE 4 Amino acid sequences of 2C3 anti-CD-52 antibodies SEQ ID NO NameAmino Acid Sequence 1 Anti-CD-52 DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSNG WTKTYLNWLLQKPGQSPQRLIYLVSKLDSGVPDR light chainFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTHL HTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2 Anti-CD-52 VQLVESGGGLVQPGGSLRLSCAASGFTFNTYW WTMNWVRQAPGKGLEWVGQIRLKSNNYATHYA heavy chainESVKGRFTISRDDSKNSLYLQMNSLKTEDTAV YYCTPVDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK 3 Anti-CD-52EVQLVESGGGLVQPGGSLRLSCAASGFTFNTY A114N WMNWVRQAPGKGLEWVGQIRLKSNNYATHYheavy chain AESVKGRFTISRDDSKNSLYLQMNSLKTEDTA

PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK 4 Anti-CD-52EVQLVESGGGLVQPGGSLRLSCAASGFTFNTY Y436S WMNWVRQAPGKGLEWVGQIRLKSNNYATHYheavy chain AESVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCTPVDFWGQGTTVTVSSASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN

5 Anti-CD-52 EVQLVESGGGLVQPGGSLRLSCAASGFTFNTY S440NWMNWVRQAPGKGLEWVGQIRLKSNNYATHY heavy chainAESVKGRFTISRDDSKNSLYLQMNSLKTEDTA VYYCTPVDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN

6 Anti-CD-52 EVQLVESGGGLVQPGGSLRLSCAASGFTFNTY S442NWMNWVRQAPGKGLEWVGQIRLKSNNYATHY heavy chainAESVKGRFTISRDDSKNSLYLQMNSLKTEDTA VYYCTPVDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLUTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN

7 Anti-CD-52 EVQLVESGGGLVQPGGSLRLSCAASGFTFNTY NGTWMNWVRQAPGKGLEWVGQIRLKSNNYATHY heavy chainAESVKGRFTISRDDSKNSLYLQMNSLKTEDTA VYYCTPVDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN

8 Anti-CD-52 EVQLVESGGGLVQPGGSLRLSCAASGFTFNTY S298N/WMNWVRQAPGKGLEWVGQIRLKSNNYATHY Y300S AESVKGRFTISRDDSKNSLYLQMNSLKTEDTAheavy chain VYYCTPVDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR

SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK

The mutants and WT control were transfected into HEK293-EBNA cells in a6-well plate format. As shown in FIG. 9, the expression level was foundto be ˜0.1 μg/ml, as analyzed by SDS-PAGE and Western blot. Expressionof mutants in conditioned media was also measured by protein A captureon Biacore. Concentration was determined using the dissociation response6 minutes after injection into immobilized Protein A. CHO-produced WT2C3 serially diluted in media from 90 μg/mL to 1.5 ng/mL was used as astandard curve. Concentrations were calculated down to ˜0.2 μg/mL by acalibration curve using a 4-parameter fit. As shown in FIG. 9, relativeexpressions levels were low and generally corresponded with the Westernblot results.

1B. Verification of Hyperglycosylation

To determine whether additional glycosylation sites were introduced bymutation, 2C3 mutant and wild type proteins were treated with theuniversal deglycosylating enzyme PNGase F and protein samples wereanalyzed by SDS-PAGE and Western blot. As shown in FIG. 10, only theA114N mutant had an increased apparent molecular weight, indicating thepresence of an additional N-linked carbohydrate.

Small scale antibody preparations were produced to purify the 2C3mutants for further verification of glycosylation site introduction. Asshown in FIG. 11, it was confirmed by SDS-PAGE that only the A114Nmutant had additional glycosylation sites introduced.

1C. Binding Properties of 2C3 Anti-CD-52 Mutants

Biacore was used to compare the binding properties of the purifiedproteins. Mouse and SEC-purified human FcRn-HPC4 were immobilized on aCM5 chip via amine coupling. Each antibody was diluted to 200, 50, and10 nM and injected over the immobilized Fc receptors. Campath,CHO-produced WT 2C3, and DEPC-treated Campath were included as positiveand negative controls. As shown in FIG. 13, the Y436S mutant displayedabout a 2-fold decrease in binding to human FcRn. Interestingly, bindingof this mutant to mouse FcRn was not affected. None of the other 2C3mutations had any considerable effect on human or mouse FcRn binding.

Biacore was used to compare the antigen binding properties of thepurified proteins using the CD-52 peptide 741 Biacore binding assay.CD-52 peptide 741 and control peptide 777 were immobilized to a CM5chip. Antibodies were serially diluted 2-fold from 60 to 0.2 nM inHBS-EP and injected in duplicate for 3 min followed by a 5 mindissociation in buffer at a 50 μL/min flow-rate. GLD52 lot 17200-084 wasincluded as a control. The surface was regenerated with 1 pulse of 40 mMHCl. A 1:1 binding model was used to fit the 7.5 to 0.2 nM curves. Asshown in FIG. 16, the A114N mutant had a slightly lower CD-52 bindingaffinity while the NGT mutant had a slightly higher affinity than therest of the mutants in this assay. The CD-52 peptide 741 Biacore bindingassay was repeated with protein purified from larger scale prep. Asshown in FIG. 17, the A114N mutant exhibited CD-52 peptide binding thatwas comparable to WT 2C3.

1D. Charge Characterization of the A114N Mutant

Isoelectric focusing (IEF) was performed to characterize the charge ofthe 2C3 mutants. Purified protein was run on immobilized pH gradient(pH3-10) acrylamide (IPG) gels. As shown in FIG. 18A, A114N was found tohave more negative charges, likely due to sialic acid residues. IntactMS data confirmed the complex structure with sialic acids on A114Nmutant. In contrast, the WT 2C3 was shown to have G0F and G1F as thedominant glycosylation species (FIGS. 18C and 18D, respectively).

Example 2 Preparation of Hyperglycosylation Mutants in Several AntibodyBackbones

In addition to the 2C3 anti-CD-52 antibody, the A114N mutation wasengineered in several other antibody backbones to confirm that theunique hyperglycosylation site could be introduced into unrelated heavychain variable domain sequences. The hyperglycosylated anti-TEM1,anti-FAP, and anti-Her2 mutants are set forth in Table 5.

TABLE 5 A114N and/or S298N mutants designed in several unrelatedantibody backbones Mutation Antibody Desired benefits Applications A114Nanti-TEM1 Additional glycosylation site at 1) Control anti-FAP the elbowhinge of heavy chain 2) Aminooxy toxin anti-Her2 for site-specificcarbohydrate- conjugation via exposed mediated conjugation sialic acidor galactose group (SAM or GAM) S298N/ anti-Her2 Switch theglycosylation from 1) Aminooxy toxin T299A/ Asn297 to an engineeredconjugation via exposed Y300S Asn298. Expect solvent sialic acid orgalactose (NNAS) exposed and complex group (SAM or GAM) carbohydrates atS298N, 2) Reduced effector offering conjugation site and function meansto remove effector function A114N/ anti-Her2 Potential for increased 1)Control NNAS conjugation yield with two 2) Aminooxy toxin conjugationsites conjugation via exposed sialic acid or galactose group (SAM orGAM)2A. Creation of Anti-TEM1 and Anti-FAP Antibody HyperglycosylationMutants

The A114N mutation, designated based upon the Kabat numbering system,was introduced into the CH1 domain of anti-TEM1 and anti-FAP bymutagenic PCR. To create the full-length antibody, the mutated VH plusresidue 114 was inserted by ligation independent cloning (LIC) into thepENTR-LIC-IgG1 vector encoding antibody CH domains 1-3. Full-lengthmutants were then cloned into the pCEP4(−E+I)Dest expression vector byGateway cloning. Mutations were confirmed by DNA sequencing Amino acidsequences of the anti-TEM1 wild type and mutated heavy and light chainsare set forth in Table 6. Mutated amino acids are highlighted in grayand the consensus glycosylation target sites created by the mutation areunderlined.

TABLE 6 Amino acid sequences of anti-TEM1 and anti-FAP antibodies SEQ IDNO Name Amino Acid Sequence  9 Anti-TEM1EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYL WT lightAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSG chainSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFG (cloneQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVV #187) CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC 10 Anti-TEM1QVQLQESAPGLVKPSETLSLTCTVSGGSIRSYY WT heavyWSWIRQPPGKGLEYIGYIYYTGSAIYNPSLQSR chain VTISVDTSKNQFSLKLNSVTAADTAVYYCARE(clone GVRGASGYYYYGMDVWGQGTTVTVSSASTK #187)GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 11Anti-TEM1 QVQLQESAPGLVKPSETLSLTCTVSGGSIRSYY A114NWSWIRQPPGKGLEYIGYIYYTGSAIYNPSLQSR VTISVDTSKNQFSLKLNSVTAADTAVYYCARE

GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K*

The mutants and wild type control were transfected into HEK293-EBNAcells in a triple flask format and purified on HiTrap protein A columns(GE Healthcare Biosciences, Pittsburgh, Pa., USA). As analyzed by A280on a NanoDrop spectrophotometer, the expression of anti-FAP A114N andanti-FAP A114C was about 3 μg/ml and about 1 μg/ml, respectively. Theexpression of anti-TEM1 A114N was about 0.04 μg/ml.

2B. Verification of Hyperglycosylation

To confirm that the additional glycosylation site was introduced intothe A114N mutants, purified protein from the A114N mutants was analyzedon reducing SDS-PAGE along with wild-type control protein. Oneadditional glycosylation site would add 2000-3000 Daltons to themolecular weight of the heavy chain. As shown in FIG. 20, SDS-PAGEindicated that the anti-FAP and anti-TEM1 A114N mutants heavy chainbands had increased apparent molecular weight, consistent withsuccessful introduction of an additional glycosylation site to bothantibodies.

2C. Creation of Anti-Her2 Antibody Hyperglycosylation Mutants

The Her-2 A114N, Her-2 A114N/NNAS, and WT Her-2 antibodies were createdby ligation independent cloning. The VH domain of Herceptin wassynthesized and PCR-amplified with two LIC-compatible sets of primers,either WT or bearing the A114N mutation. To obtain a full-lengthantibody, amplified VH inserts (WT or A114N) were cloned into two pENTRvectors encoding CH 1-3 domains, pENTR-LIC-IgG1 WT and pENTR-LIC-IgG1NNAS, resulting in three full-length mutants (A114N, NNAS, A114N/NNAS)and WT control as entry clones on pENTR. These mutants were cloned intothe pCEP4(−E+I)Dest expression vector, by Gateway cloning. Mutationswere confirmed by DNA sequencing. Amino acid sequences of the anti-Her-2wild type and mutated heavy and light chains are set forth in Table 7.Mutated amino acids are highlighted in gray and the consensusglycosylation target sites created by the mutation are underlined

TABLE 7 Amino acid sequences of anti-Her-2 antibodies SEQ ID NO NameAmino Acid Sequence 12 Anti-Her-2 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAV WTAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSR light chainSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 13 Anti-Her-2 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTY WTIHWVRQAPGKGLEWVARIYPTNGYTRYADSV heavy chainKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC SRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 14Anti-Her-2 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTY A114NIHWVRQAPGKGLEWVARIYPTNGYTRYADSV heavy chainKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC

VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 15Anti-Her2 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTY NNASIHWVRQAPGKGLEWVARIYPTNGYTRYADSV heavy chainKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC SRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA

YKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 16Anti-Her2 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTY A114N/IHWVRQAPGKGLEWVARIYPTNGYTRYADSV NNAS KGRFTISADTSKNTAYLQMNSLRAEDTAVYYCheavy chain

VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA

YKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK2D. Expression of the A114N Anti-Her2 Antibody Hyperglycosylation Mutant

The A114N anti-Her2 and wild type constructs were transfected withLipofectamine-2000 (2.5:1 ratio of reagent to DNA) and XtremeGene HP(3:1 ratior of reagent to DNA) into HEK293-EBNA cells in 12 tripleflasks. Octet measurement of aliquots from day 3 conditioned media (CM)showed that protein expression was consistent across 6 flasks for bothLipofectamine-2000 and XtremeGene HP. As shown in Table 8, the overalltransfection efficiency was about 30% higher with XtremeGene HP.Conditioned media collected on day 3 was pooled together for bothtransfection conditions and purified by protein A column. Octetmeasurement showed 1.8 ug/ml antibody in the serum-containing mock mediaversus 0 ug/ml in no serum mock media.

TABLE 8 A114N anti-Her2 hyperglycosylation mutant expressionLipofectamine- XtremeGene 2000 HP Purified protein Concentration 1.723.18 from protein A (mg/ml) column Volume (ml) 3.5 3.5 Total protein(mg) 6.02 11.13 Buffer-exchanged Concentration 15.59 16.86 protein(mg/ml) Volume (ml) 0.2 0.36 Total protein (mg) 3.1 6.07 % Recovery 51.854.5

Conditioned media from Day 6 was collected and purified separately foreach transfection condition. Both eluates were buffer-exchangedseparately into PBS, pH 7.2, and concentrated ˜15-fold using Amicon-4(50 kD cut-off) columns. Day 6 CM showed higher expression levelcompared to Day 3 CM. As shown in Table 8, a total of 3 mg of HerceptinA114N 15.59 mg/ml (from Lipofectamine transfection) and 6 mg ofHerceptin A114N 16.86 mg/ml (from XtremeGene HP transfection) wasproduced from day 6 conditioned media for additional downstreamapplications, such as antibody-drug conjugation.

2E. SDS-PAGE and HIC Analyis of the A114N Anti-Her2 Mutant

Prior to conjugation, purified A114N Herceptin was characterized bySDS-PAGE and HIC (hydrophobic interaction chromatography). As shown inFIG. 21, the quality of purified A114N Herceptin was determined to besuitable for further downstream applications.

2F. Conjugation to Engineered Glycosylation

It was demonstrated that: a) a glycosylation site was introduced atKabat position 114 site on anti-TEM1; b) the A114N mutant hadhyperglycosylation on the heavy chain by reducing SDS-PAGE; and c) theA114N hyperglycosylated mutant had complex carbohydrate structure byintact LC/MS, including terminal sialic acids and galactose, which areideal for SAM and GAM conjugation. To confirm that the engineeredglycosylation site was suitable for conjugation, anti-TEM1 A114N wasconjugated with a 5 kDa PEG via aminooxy chemistry. As shown in FIG. 22,PEG was successfully conjugated to anti-TEM1 A114N through an aminooxylinkage. This mutant was also successfully prepared on the anti-FAP andanti-CD-52 2C3 backbones (not shown). These data demonstrate that theglycosylation site at N114 is useful for conjugation of effectormoieties.

Example 3 Generation of S298N/Y300S Fc Mutants

Engineered Fc variants was designed and generated in which a newglycosylation site was introduced at EU position Ser 298, next to thenaturally-occurring Asn297 site. The glycosylation at Asn297 was eithermaintained or ablated by mutation. Mutations and desired glycosylationresults are set forth in Table 9.

TABLE 9 Glycosylation states of various antibody variants # MutationDesired Glycosylation State Applications 17 N297Q No glycosylation(agly) Agly Control 18 T299A No glycosylation (agly) Agly Control,unknown effector function 19 N297Q/S298N/Y300S No glycosylation at 297but Reduced effector (NSY) engineered glycosylation site at function;Conjugation 298 via exposed sialic acid or galactose groups. 20S298N/T299A/Y300S No glycosylation at 297 but Reduced effector (STY)engineered glycosylation site at function; Conjugation 298 via exposedsialic acid or galactose groups. 21 S298N/Y300S (SY) Two potentialglycosylation Reduced effector sites at 297 & 298; Alterations function;Conjugation in glycosylation pattern. via exposed sialic acid orgalactose groups. 22 Wild-type 297 control3A. Creation of H66 αβ-TCR Antibody Altered Glycosylation Variants

Mutations were made on the heavy chain of αβ T-cell receptor antibodyclone #66 by Quikchange using a pENTR_LIC_IgG1 template. The VH domainof HEBE1 Δab IgG1 #66 was amplified with LIC primers before being clonedinto mutated or wild type pENTR_LIC_IgG1 by LIC to create full-lengthmutant or wild-type antibodies. The subcloning was verified withDraIII/XhoI double digest, producing an approximately 1250 bp-sizedinsert in the successful clones. Those full-length mutants were thencloned into an expression vector, pCEP4(−E+I)Dest, via Gateway cloning.The mutations were confirmed by DNA sequencing Amino acid sequences ofthe WT H66 anti-αβTCR heavy and light chains and the mutated H66 heavychains are set forth in Table 10. Mutated amino acids are highlighted ingray and the consensus glycosylation target sites created by themutation are underlined.

TABLE 10 Amino acid sequences of H66 anti-αβTCR antibodies SEQ ID NOName Amino Acid Sequence 23 Anti-αβTCREIVLTQSPATLSLSPGERATLSCSATSSVSYMHW clone H66YQQKPGQAPRRLIYDTSKLASGVPARFSGSGSGT lightSYTLTISSLEPEDFAVYYCQQWSSNPLTFGGGTK chainVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC* 24 Anti-αβTCREVQLLQSGGGLVQPGGSLRLSCAASGYKFTSYV clone H66MHWVRQAPGKGLEWVGYINPYNDVTKYNEKFK heavy GRFTLSRDNSKNTLYLQMNSLRAEDTAVYYCARchain GSYYDYDGFVYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK* 25 Anti-αβTCREVQLLQSGGGLVQPGGSLRLSCAASGYKFTSYV clone H66MHWVRQAPGKGLEWVGYINPYNDVTKYNEKFK S298N/GRFTLSRDNSKNTLYLQMNSLRAEDTAVYYCAR Y300SGSYYDYDGFVYWGQGTLVTVSSASTKGPSVFPL heavyAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG chainALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN

APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK* 26 Anti-αβTCREVQLLQSGGGLVQPGGSLRLSCAASGYKFTSYV clone H66MHWVRQAPGKGLEWVGYINPYNDVTKYNEKFK S298N/GRFTLSRDNSKNTLYLQMNSLRAEDTAVYYCAR T299A/GSYYDYDGFVYWGQGTLVTVSSASTKGPSVFPL Y300SAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG heavyALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ chainTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN

APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK* 27 Anti-αβTCREVQLLQSGGGLVQPGGSLRLSCAASGYKFTSYV clone H66MHWVRQAPGKGLEWVGYINPYNDVTKYNEKFK N297Q/GRFTLSRDNSKNTLYLQMNSLRAEDTAVYYCAR S298N/GSYYDYDGFVYWGQGTLVTVSSASTKGPSVFPL Y300SAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG heavyALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ chainTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVD

APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK*

The mutant, wild-type, and two aglycosylated control (HEBE1 Agly IgG4and HEBE1 Δab IgG1 in pCEP4) constructs were transfected intoHEK293-EBNA cells in triple-flasks for expression. Proteins werepurified from 160 ml of conditioned media (CM) with 1 ml HiTrap proteinA columns (GE) using a multi-channel peristaltic pump. Five microgramsof each resulting supernatant were analyzed on 4-20% Tris-Glycinereducing and non-reducing SDS-PAGE gels (see FIG. 2). The heavy chainsof the aglycosylated mutants (N297Q, T299A, and Agly controls), havemigrated further (arrowhead), consistent with the loss of the glycans inthese antibodies. The heavy chains of the engineered glycosylatedantibodies (NSY, STY, SY, Δab, and wt control, arrows), however, migratesimilarly to the wild-type control. This result is consistent with theexistence of an engineered glycosylation site at EU position 298.SEC-HPLC analysis indicated that all mutants are expressed as monomers.

3B. Glycosylation Analysis by LC-MS

The engineered H66 IgG1 Fc variants were partially reduced with 20 mMDTT at 37° C. for 30 min. The samples were then analyzed by capillaryLC/MS on an Agilent 1100 capillary HPLC system coupled with a QSTAR qqTOF hybrid system (Applied Biosystems).

A Bayesian protein reconstruction with baseline correction and computermodeling in Analyst QS 1.1 (Applied Bisoystem) was used for dataanalysis. In the S298N/T299A/Y300S H66 antibody mutant, oneglycosylation site was observed at amino acid 298 with bi-antennary andtri-antennary complex-type glycans detected as the major speciesalongside G0F, G1F and G2F (see FIG. 34). This altered glycosylationprofile is consistent which shifted glycosylation at N298 instead of thewild-type glycosylation site at N297.

3C. Binding Properties of αβTCR Antibody Mutants to Human FcγRIIIa andFcγRI Using Biacore

Biacore was used to assess binding to recombinant human FcγRIIIa (V158 &F158) and FcγRI. All four flowcells of a CM5 chip were immobilized withanti-HPC4 antibody via the standard amine coupling procedure provided byBiacore. The anti-HPC4 antibody was diluted to 50 μg/mL in 10 mM sodiumacetate pH 5.0 for the coupling reaction and injected for 25 min at 5μL/min Approximately 12,000 RU of antibody was immobilized to the chipsurface. Recombinant human FcγRIIIa-V158 and FcγRIIIa-F158 were dilutedto 0.6 μg/mL in binding buffer (HBS-P with 1 mM CaCl₂) and injected toflowcells 2 and 4, respectively, for 3 min at 5 μL/min to capture300-400 RU receptor on the anti-HPC4 chip. In order to distinguishbetween the low binders, three times more rhFcγRIIIa was captured on theanti-HPC4 surface than usually used in this assay. Flowcells 1 and 3were used as reference controls. Each antibody was diluted to 200 nM inbinding buffer and injected over all four flowcells for 4 min, followedby 5 min dissociation in buffer. The surfaces were regenerated with 10mM EDTA in HBS-EP buffer for 3 min at 20 μL/min. The results of theseexperiments are shown in FIG. 3.

Biacore was also used to compare the FcγRI binding. Anti-tetra Hisantibody was buffer exchanged into 10 mM sodium acetate pH 4.0 using aZeba Desalting column and diluted to 25 μg/mL in the acetate buffer foramine coupling. Two flowcells of a CM5 chip were immobilized with ˜9000RU of the anti-Tetra-His antibody after 20 min injection at 5 μL/min. Asin the previous experiment, ten times more FcγRI was captured to theanti-tetra-His surface in order to compare samples with weak binding.Recombinant human FcγRI was diluted 10 μg/mL in HBS-EP binding bufferand injected to flowcell 2 for 1 min at 5 μL/min to capture ˜1000 RUreceptor to the anti-tetra-His chip. A single concentration of antibody,100 nM, was injected for 3 min at 30 μL/min over the captured receptorand control surface. Subsequently, dissociation was monitored for threeminutes. The surface was then regenerated with two 30 second injectionsof 10 mM glycine pH 2.5 at 20 μL/min. The results of these experimentsare shown in FIG. 4.

These results demonstrate a striking decrease in binding of theglycoengineered mutants to FcγRIIIa or FcγRI. H66 S298N/T299A/Y300S inparticular has almost completely abolished binding to both receptors.This mutant was chosen for more detailed analysis.

3D. Stability Characterization Using Circular Dichroism (CD)

The stability of the S298N/T299A/Y300S antibody mutant was monitored bya Far-UV CD thermo melting experiment in which the CD signal at 216 nmand 222 nm was monitored as increasing temperature lead to the unfoldingof the antibody (denaturation).

Temperature was controlled by a thermoelectric peltier (Jasco modelAWC100) and was increased at a rate of rC/min from 25-89° C. The CDspectra were collected on a Jasco 815 spectrophotometer at a proteinconcentration of approximately 0.5 mg/mL in PBS buffer in a quartzcuvette (Hellma, Inc) with a path length of 10 mm. The scanning speedwas 50 nm/min and a data pitch of 0.5 nm. A bandwidth of 2.5 nm was usedwith a sensitivity setting of medium. The CD signal and HT voltage werecollected from 210-260 nm with data intervals of 0.5 nm and attemperature intervals of 1° C. and four replicate scans were performedfor each sample. The results demonstrate that both delta AB H66 and theS298N/T299A/Y300S H66 mutant exhbit similar thermal behaviors and haveapproximately the same onset temperature for degradation (around 63° C.)(FIG. 35), further suggesting that they have comparable stability.

Example 4 Functional Analysis of Fc-engineered Mutants

Fc-engineered mutants were assessed through a PBMC proliferation assayand a cytokine release assay. In the PBMC proliferation assay, humanPBMC were cultured with increasing concentrations of therapeuticantibody for 72 hours, ³H-thymidine was added and cells were harvested18 hours later. For the T cell depletion/Cytokine Release assay, humanPBMC were cultured with increasing concentrations of therapeuticantibody and were analyzed daily for cell counts and viability (Vi-Cell,Beckman Coulter) out to day 7. Cell supernatants were also harvested,stored at −20° C. and analyzed on an 8-plex cytokine panel (Bio-Rad).

Normal donor PBMC were thawed and treated under the following conditions(all in media containing complement): Untreated; BMA031, moIgG2b 10ug/ml; OKT3, moIgG2a 10 ug/ml; H66, huIgG1 deltaAB 10 ug/ml, 1 ug/ml and0.1 ug/ml; H66, huIgG1 S298N/T299A/Y300S 10 ug/ml, 1 ug/ml and 0.1ug/ml.

Cytokines were harvested at day 2 (D2) and day 4 (D4) for BioplexAnalysis (IL2, IL4, IL6, IL8, IL10, GM-CSF, IFNg, TNFa). Cells werestained at D4 for CD4, CD8, CD25 and αβTCR expression.

The results, shown in FIGS. 5-8, demonstrate that H66 S298N/T299A/Y300Sbehaved similarly to the H66 deltaAB in all the cell based assaysperformed, showing minimal T-cell activation by CD25 expression, bindingto αβTCR (with slightly different kinetics to deltaAB), and minimalcytokine release at both D2 and D4 time points. The S298N/T299A/Y300Smutant thus eliminated effector function as effectively as the deltaABmutation.

Example 5 Preparation and Characterization of an Engineered Fc Variantin the Anti-CD52 Antibody Backbone

In addition to the H66 anti-αβTCR antibody, the S298N/Y300S mutation wasalso engineered in an anti-CD52 antibody backbone (clone 2C3). Thismutant was then examined in order to determine whether the observedeffector function modulation seen in the S298N/Y300S H66 anti-αTCRantibody was consistent in another antibody backbone.

5A. Creation of 2C3 Anti-CD52 Antibody Altered Glycosylation Variants

First, S298N/Y300S 2C3 variant DNA was prepared by quick changemutagenesis using pENTR_LIC_IgG1, and WT 2C3 VH was cloned into themutated vector by LIC. Full-length mutants were cloned into the pCEP4(−E+I)Dest expression vector using Gateway technology. Mutations weresubsequently confirmed by DNA sequencing and the sequences are set forthin Table 11. The mutants were then transfected into HEK293-EBNA cells ina 6-well plate format and the protein was purified from conditionedmedia. Anti-CD52 2C3 wild-type antibody was produced in parallel as acontrol. The expression level was found to be 0.1 μg/mL using SD-PAGEand Western blot analyses (FIG. 9A). Expression of mutants in neatconditioned media was also measured by protein A capture on Biacore.Concentration was determined using the dissociation response after asix-minute injection to immobilized protein A. CHO-produced WT 2C3serially diluted in media from 90 μg/mL to 1.5 ng/mL was used as astandard curve. Concentrations were calculated within approximately 0.2μg/mL by a calibration curve using a 4-parameter fit. Relativeexpression levels were low and generally agree with the Western blotdata (FIG. 9B).

TABLE 11 Anti-CD52 clone 2C3 antibody sequences SEQ ID NO NameAmino Acid Sequence 28 Anti- DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSNGKTYLCD-52 NWLLQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSGTD 2C3 WTFTLKISRVEAEDVGVYYCVQGTHLHTFGQGTRLEIKR lightTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK chainVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*29 Anti- EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMN CD-52WVRQAPGKGLEWVGQIRLKSNNYATHYAESVKGRF 2C3 WTTISRDDSKNSLYLQMNSLKTEDTAVYYCTPVDFWGQ heavyGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV chainKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK* 30 Anti-EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMN CD-52WVRQAPGKGLEWVGQIRLKSNNYATHYAESVKGRF 2C3TISRDDSKNSLYLQMNSLKTEDTAVYYCTPVDFWGQ S298N/GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV Y300SKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS heavySVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC chainDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE

PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK*5B. Glycosylation Analysis Using PNGaseF

To evaluate the additional glycosylation sites introduced by themutation, the enriched S298N/Y300S mutant was de-glycosylated withPNGase F. It did not demonstrate any apparent change in molecularweight, which indicates that no additional carbohydrate was present(FIG. 10). Small scale preparations were performed in order to purifythese mutants for further characterization and the results reconfirmedthat there was not an additional carbohydrate present on the S298N/Y300Smutant (FIG. 11).

5C. Binding Properties of 2C3 Anti-CD52 Antibody Mutants to HumanFcγRIIIa Using Biacore

Biacore was also used to characterize the antigen-binding, FcγRIII, andbinding properties of the purified antibodies (see FIGS. 12, 13, and14). The S298N/Y300S 2C3 variant bound to the CD52 peptide tightly andthe binding sensorgram was undistinguishable from the wild-type control,demonstrating that this mutation does not affect its antigen binding(FIG. 12A).

To assay for Fc effector function, FcγRIII receptor (Val158) was used inbinding studies. The mutant and wild-type control antibody were dilutedto 200 nM and injected to HPC4-tag captured FcγRIIIa. FcγRIII bindingwas almost undetectable for the S298N/Y300S mutant, which indicated aloss of effector function by this variant (FIG. 12B and FIG. 14A). Tofurther assay for Fc effector function, the FcγRIII receptor (Phe158)was also used in binding studies. The mutant and wild-type controlantibodies were diluted to 200 nM and injected to HPC4-tag capturedFcγRIIIa. FcγRIII binding was almost undetectable for the S298N/Y300Smutant, which indicates a loss of effector function with the Phe158variant (FIG. 14B). Finally, Biacore was used to compare the FcRnbinding properties of the purified proteins. Mouse and SEC-purifiedhuman FcRn-HPC4 were immobilized to a CM5 chip via amine coupling. Eachantibody was diluted to 200, 50, and 10 nM and injected over thereceptors. Campath, CHO-produced WT 2C3, and DEPC-treated Campath wereincluded as positive and negative controls. These data show that themutant binds to both human and murine FcRn receptor with the sameaffinity as the wild-type antibody control and that it likely has noalterations in its circulation half-life or other pharmacokineticproperties. (see FIG. 12C, FIGS. 13A and B). Accordingly, theS298N/Y300S mutation is applicable to antibodies in general, to reduceor eliminate undesired Fc effector function, for example throughengagement of human Fcγ receptors.

Example 6 Circulating Immune Complex Detection in the S298N/Y300S Mutant

Circulating immune complex detection was also investigated using a C1qbinding assay for the S298N/Y300S mutant and WT control. High bindingCostar 96-well plates were coated overnight at 4° C. with 100 μl of2-fold serially diluted 2C3 Abs at concentrations ranging from 10-0.001μg/ml in coating buffer (0.1M NaCHO₃ pH 9.2). ELISA analysis showed thatC1q binding is reduced for the S298N/Y300S mutant compared to WT (FIG.15A). The binding of anti-Fab Ab to the coated 2C3 Abs confirmedequivalent coating of the wells (FIG. 15B).

Example 7 Separation and Analysis of S298N/Y300S Mutant UsingIsoelectric Focusing

A pH 3-10 Isoelectric Focusing (IEF) gel was run to characterize theS298N/Y300S mutants. 5298/Y300S was found to have more negative charges,and therefore, likely more sialic acid molecules (FIG. 18A). Both theS298N/Y300S mutant and WT 2C3 were shown by intact MS to have G0F andG1F as the dominant glycosylation species (FIGS. 18 B and D,respectively).

Example 8 Antigen Binding Affinity of S298N/Y300S

Biacore was used to compare the antigen binding affinity of WT anti-CD522C3 Ab and the S298N/Y300S mutant that had been prepared and purifiedfrom both smaller (FIG. 16) and larger (FIG. 17) scale expressions. CM5chips immobilized with CD52 peptide 741 and control peptide 777 wereobtained. Antibodies were serially diluted 2-fold from 60 to 0.2 nM inHBS-EP and were then injected over the chip surface for 3 min followedby a 5 min dissociation in buffer at a flow rate of 50 μl/min. Thesurface was then regenerated with a pulse of 40 mM HCl. These analyseswere performed in duplicate and demonstrate that the S298N/Y300S mutantand WT 2C3 antibodies show comparable CD52 peptide binding.

A media screening platform was designed to test functional bindingproperties prior to purification in order to screen antibodies createdduring small scale transfections. These tests were performed using Octet(FIG. 19A) to determine concentration and used Protein A biosensors anda GLD52 standard curve. Samples were diluted to 7.5 and 2 nM in HBS-Epfor a CD52 binding comparison using Biacore (FIG. 19B). The results ofthe peptide binding assay showed that both the S298N/Y300S mutant andthe WT 2C3 antibodies have comparable CD52 peptide binding. Furthermore,these analyses demonstrate that Octet and Biacore work well to predictantigen binding by antibodies from small scale transfections.

Example 9 Preparation of S298N/Y300S, S298N/T299A/Y300S, andN297Q/S298N/Y300S Altered Glycosylation Mutants in Additional AntibodyBackbones

In addition to the anti-αβ-TCR antibody and 2C3 anti-CD-52 antibody, the5298/Y300S, S298N/T299A/Y300S, and N297Q/S298N/Y300S mutations wereengineered in other antibody backbones to confirm that the additionaltandem glycosylation site could be introduced into unrelated heavy chainvariable domain sequences. The alternatively glycosylated anti-CD-5212G6 and anti-Her2 mutants are set forth in Tables 12 and 13.

TABLE 12 Anti-CD52 clone 12G6 antibody sequences SEQ ID NO NameAmino Acid Sequence 31 Anti- DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSNGKTYLCD-52 NWVLQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSGTD 12G6FTLKISRVEAEDVGVYYCVQGSHFHTFGQGTKLEIKR WTTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV lightQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK chainADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 32 Anti-EVQLVESGGGLVQPGGSLRLSCAASGFPFSNYWMNW CD-52VRQAPGKGLEWVGQIRLKSNNYATHYAESVKGRFTIS 12G6RDDSKNSLYLQMNSLKTEDTAVYYCTPIDYWGQGTT WTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF heavyPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT chainVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* 33 Anti-EVQLVESGGGLVQPGGSLRLSCAASGFPFSNYWMNW CD-52VRQAPGKGLEWVGQIRLKSNNYATHYAESVKGRFTIS 12G6RDDSKNSLYLQMNSLKTEDTAVYYCTPIDYWGQGTT S298N/VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF Y300SPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT heavyVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH chainTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV

TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K* 34 Anti-EVQLVESGGGLVQPGGSLRLSCAASGFPFSNYWMNW CD-52VRQAPGKGLEWVGQIRLKSNNYATHYAESVKGRFTIS 12G6RDDSKNSLYLQMNSLKTEDTAVYYCTPIDYWGQGTT S298N/VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF T299A/PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT Y3005VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH heavyTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV chain

TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K* 35 Anti-EVQLVESGGGLVQPGGSLRLSCAASGFPFSNYWMNW CD-52VRQAPGKGLEWVGQIRLKSNNYATHYAESVKGRFTIS 12G6RDDSKNSLYLQMNSLKTEDTAVYYCTPIDYWGQGTT N297Q/VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF S298N/PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT Y300SVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH heavyTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV chain

TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K*

TABLE 13 Anti-Her2 antibody sequences SEQ ID NO Name Amino Acid Sequence36 Anti- DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQ Her2QKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTI WTSSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAP lightSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV chainDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC* 37Anti- EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWV Her2RQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADT WTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDY heavyWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG chainCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK* 38 Anti-EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWV Her2RQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADT S298N/SKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDY T299A/WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG Y300SCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY heavySLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK chainSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR

LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK*

Example 10 Generation of Altered Antibodies Containing Reactive GlycanMoieties

In order to generate antibodies containing glycan moieties capable ofreacting with derivatized effector moieties, an anti-HER antibody wasfirst glycosylated in vitro using glycosyltransferase and relevant UDPsugar donors. For example, to introduce the sialic acid residues, donorantibodies were first galactosylated with β-galactosyltransferase,followed with sialylation with α2,6-sialyltransferase according to themethods of Kaneko et al. (Kaneko, Y., Nimmerjahn, F., and Ravetch, J. V.(2006) Anti-inflammatory activity of immunoglobulin G resulting from Fcsialylation. Science 313, 670-3). The reaction was performed in aone-pot synthesis step using β-galactosyltransferase (50 mU/mg, Sigma)and α2,6-sialyltranafrease (5 ug/mg, R&D system) with donor sugarnucleotide substrates, UDP-galactose (10 mM) and CMP-sialic acid (10 mM)in 50 mM MES buffer (pH 6.5) containing 5 mM MnCl₂. The reaction mixturecontaining 5 mg/ml anti-HER2 antibody was incubated for 48 hours at 37°C. The sialylation was verified using MALDI-TOF MS analysis ofpermethylated glycans released from the antibody with PNGase F, sialicacid content analysis using Dionex HPLC and lectin blotting with SNA, alectin specific for α2,6-sialic acid.

MALDI-TOF analysis of glycans released by PNGase F treatment of thesialylated anti-HER2 antibody indicated that native glycans had beencompletely remodeled with a mainly monosialylated biantennary structure,A1F (FIG. 27A) together with small amount of disialylated species.Treatment of the antibody with higher amounts of α2,6-sialyltransferaseproduced more homogenous populations of the A1F glycoform, suggestingthat either the enzyme activity or glycan localization may prevent fullsialylation. Sialic acid content was determined to be ˜2 mol per mol ofantibody, which is consistent with A1F glycan as the major glycoformspecies (FIG. 27B). Lectin blotting with a SAN lectin, Sambucus nigraagglutinin specific for α2,6-linked sialic acid, confirmed that thesialic acid was present in an α2,6-linkage configuration (FIG. 27C).

In conclusion, although the native protein glycans are somewhatheterogeneous, remodeling through galactosyl and sialyltransferasesyields a nearly homogeneous antibody with monosialylated but fullygalactosylated biantennary glycans (A1F). The introduction of only ˜1sialic acid on the two galactose acceptors on each branched glycan maybe due to limited accessibility of one of the galactoses from glycanswhich are often buried in the antibody or non-covalent interactions ofthe glycans with the protein surface.

Example 11 Oxidation of Altered Antibodies Containing Reactive GlycanMoieties

Once the sialylation was verified, the in-process oxidation ofsialylated anti-HER2 antibody with various concentrations of periodate(0.25 to 2 mM) was investigated. The sialylated antibody was firstbuffer-exchanged into 25 mM Tris-HCl (pH 7.5) containing 5 mM EDTAfollowed by buffer exchange with PBS buffer. The buffered antibodymixture was then applied to protein A Sepharose column pre-equilibratedwith PBS buffer. After the column was washed with 15 column volumes ofPBS, 15 column volumes of PBS containing 5 mM EDTA, and 30 columnvolumes of PBS, it was then eluted with 25 mM citrate phosphate buffer(pH 2.9). The eluates were immediately neutralized with dibasicphosphate buffer and the antibody concentrated using Amicon ultra fromMillipore. Following purification, the sialylated anti-HER2 antibodythen was oxidized with sodium periodate (Sigma) in 100 mM sodium acetatebuffer (pH 5.6) on ice in the dark for 30 minutes, and the reactionquenched with 3% glycerol on ice for 15 minutes. The product wasdesalted and exchanged into 100 mM sodium acetate (pH 5.6) by 5 roundsof ultrafiltration over 50 kDa Amicons. FIG. 28A shows sialic acidcontent analysis of sialylated antibody titrated with various amounts ofperiodate. Complete oxidation of the sialic acid residues was achievedat a periodate concentration above 0.5 mM. Indeed, a periodateconcentration as low as 0.5 mM was enough to fully oxidize theintroduced sialic acid. Accordingly, a 1 mM concentration of periodatewas chosen for oxidation of sialylated antibody for drug conjugation.

Oxidation can have adverse effects on the integrity of an antibody. For,the oxidation of methionine residues, including Met-252 and Met-428,located in Fc CH3 region, close to FcRn binding site are known to affectFcRn binding which is critical for prolonging antibody serum half-life(Wang, W., et al. (2011) Impact of methionine oxidation in human IgG1 Fcon serum half-life of monoclonal antibodies. Mol Immunol 48, 860-6).Accordingly, to examine the potential side effects of periodateoxidation on methionine residues (e.g., Met-252) critical for FcRninteraction, the oxidation state of the sialylated antibody wasdetermined by LC/MS analysis of a trypsin peptide digest. This analysisrevealed ˜30% oxidation of Met-252 and <10% oxidation of Met-428 aftertreatment of the sialylated trastuzumab with 1 mM periodate. Todetermine the impact of this degree of methionine oxidation on FcRnbinding, the FcRn binding kinetics for each antibody was evaluated usingsurface plasmon resonance (BIACORE). This analysis revealed thatoxidation state correlated with a minor loss in FcRn binding (12% and26% reduction in Ka for mouse and human FcRn, see FIGS. 28B and 28Crespectively). Notably, a ˜25% reduction in the Ka for human FcRn hasbeen reported to have no effect on the serum half-life in a human FcRntransgenic mouse, since a single intact FcRn site on each antibody issufficient to provide functionality and the PK advantage (Wang et al.,Id).

In summary, these data indicate that the introduction ofperiodate-sensitive sialic acid residues by sialyltransferase treatmentpermits the use of much lower concentrations of periodate, resulting inminimal side effects on antibody-FcRn interactions and antibodyintegrity as assessed by aggregation (≤1%). Thus, the use of sialylatedantibodies according to the methods of the invention provides a widerwindow of oxidation conditions to be employed, allowing the reproduciblegeneration of active glycoconjugates without an effect on serumhalf-life.

Example 12 Synthesis of Reactive Effector Moieties

In order to facilitate conjugation with the aldehyde-derivatizedantibody glycoforms of the invention, candidate drug effector moieties(e.g., Momomethyl Auristatin E (MMAE) and Dolastatin 10 (Do110)) werederivatized with aminooxy-cystamide to contain functional groups (e.g.,aminooxy-cys) specifically reactive with the aldehyde.

Briefly, to generate aminooxy-cystamide as a starting material,S-Trityl-L-cysteinamide (362 mg, 1 mmol) was added to a 3 mL of a DMFsolution of t-BOC-aminooxyacetic acid N-hydroxysuccinimide ester (289mg, 1 mmol). The reaction was complete after 3 h as evident from HPLCanalysis. The reaction mixture was subsequently diluted with 30 ml ofdichloromethane and was washed with 0.1 M sodium bicarbonate solution(2×20 mL), water (2×20 mL), and brine (2×20 mL). The solution was driedover anhydrous sodium sulfate, filtered and concentrated to dryness. Tothis dried residue was added 3 mL of TFA followed by 150 μL oftriethylsilane. The resulting solution was precipitated from t-butylmethyl ether and the process repeated three times. After filtration, theresidue was dried under reduced pressure yielding 205 mg of an off whitesolid (67% yield). The compound was used for next step without furtherpurification.

To generate aminooxy-derivatized MMAE (Aminooxy-Cys-MC-VC-PABC-MMAE),30.1 mg of aminooxy-cystamide (0.098 mmol, 2 eq.) was combined with 64.6mg of MC-VC-PABC-MMAE (0.049 mmol), and 100 μL of triethylamine in 3 mLof DMF. The resulting reaction mixture was stirred at room temperaturefor 15 minutes, by which time reaction was complete according to HPLCanalysis. The compound was purified by preparative HPLC yielding 45 mg(62%) of the desired product as an off-white solid. Reversed-phase HPLCanalysis suggested the purity of the compound to be >96%. ESI calcd forC73H116N14018S (MH)⁺1509.8501; found, m/z 1509.8469.

To generate aminooxy-derivatized Do110(Aminooxy-Cys-MC-VC-PABC-PEG8-Do110), 7.4 mg (0.024 mmol, 3 eq.) ofaminooxy-cystamide, 12 mg (0.008 mmol) of MC-VC-PABC-PEG8-Do110 and 30μL triethylamine were combined in in 3 mL of DMF. The reaction wascomplete within 15 minutes according to HPLC analysis. Preparative HPLCpurification resulted in 6.2 mg (46%) of the desired product as anoffwhite solid. Reversed-phase HPLC analysis suggests the purity of thecompound to be >96%. ESI calcd for C80H124N16O19S2 (MH)⁺1678.0664;found, m/z 1678.0613.

Example 13 Sialic Acid-Mediated (SAM) Conjugation of Reactive EffectorMoieties

Following desalting, drug-linkers of Example 11 were combined with theoxidized, sialylated antibodies of Example 10 in 75% DMSO at aconcentration of 25 mM to achieve a 24:1 molar ratio of drug-linker toantibody and a final antibody concentration at 5 mg/ml. The mixture wasincubated overnight at room temperature. The unincorporated drug-linkersand any free drugs were scavenged using BioBeads. The product wasbuffer-exchanged into Histidine-Tween buffer using PD-10 columns andsterile filtered. The endotoxin levels were determined and less than 0.1EU/mg ADC was achieved for in vivo study.

FIG. 29A-C shows a hydrophobic interaction chromatograph (HIC) ofdifferent sialylated antibodies (anti FAP B11 and G11 and the anti-HER2antibody of Example 11) glycoconjugated to AO-MMAE. Sialylated HER2antibody was also conjugated with the drug-linker,AO-Cys-MC-VC-PABC-PEGS-Do110 (FIG. 29D). This analysis reveals thatthere are mainly one or two drug conjugates per antibody with adrug-to-antibody ratio (DAR) ranging from 1.3-1.9. The increasedretention time of the Do110 glycoconjugate (FIG. 29D) as compared to theMMAE glycoconjugate (FIG. 29C) is likely due to the greaterhydrophobicity of Do110.

LC-MS analysis was also conducted with an anti-HER antibody conjugatedwith two different drug-linkers (AO-MMAE or AO-PEGS-Do110) at 30 mgscale. This analysis showed similar DAR values of 1.7 and 1.5 followingconjugation, which is comparable to HIC analysis. Size-exclusionchromatograpy (SEC) showed very low levels (1%) of aggregates in theseconjugates.

In addition to native (wild-type) sialylated antibodies,glycoconjugation of effector moieties to sialylated antibodiescontaining non-native (engineered) glycan N-linked to engineeredglycosylation sites was also investigated. FIG. 30B shows a HICchromatograph of a sialylated antibody successfully conjugated to anaminooxy-derivatized MMAE toxin (AO-MMAE; FIG. 30A) via an engineeredglycan present at the engineered glycosylation site A114N. Approximately60% of the sialylated antibody was successfully glycoconjugated to MMAEcytotoxin. Accordingly, this analysis confirms that the glycoconjugationmethods of the invention may be applied to both wild-type and engineeredglycans of a binding polypeptide.

Example 14 In Vitro ADC Cell Proliferation Assays

The in vitro activity of the anti-HER and anti-FAP glycoconjugatemolecules of the invention were also compared with corresponding thiolconjugates containing the same drug moiety linked via thiol linkages tohinge region cysteines of the same donor antibody. The thiol conjugatescontained approximately twice the number of drugs per antibody (DAR)than the glycoconjugates. Thiol-based conjugation was performed asdescribed by Stefano et al (Methods in Molecular Biology 2013, inpress). Her2+ SK-BR-3 and Her2− MDA-MB-231 cell lines were then employedto evaluate the relative efficacy of each ADC. The results of thisanalysis are presented in Table 15 below.

TABLE 15 EC₅₀ comparison of glycoconjugates and thiol conjugates DAREC₅₀ (ng/ml) Anti-HER-MC-VC-PABC-MMAE 3.8* 2.3 (Thiol MMAE)AntiHER-AO-Cys-MC-VC-PABC-MMAE 1.7* 4.7 (Glyco MMAE)Anti-HER-MC-VC-PABC-PEG8-Dol10 3.9* 0.45 (Thiol Dol10)Anti-HER-AO-Cys-MC-VC-PABC-PEG8- 1.5* 0.97 Dol10 (Glyco Dol10) Anti FAPB11-MC-VC-PABC-MMAE 3.3** 382.4 (Thiol MMAE), CHO + FAP Anti FAPB11-AO-Cys-MC-VC-PABC- 1.5** 682.4 MMAE (Glyco MMAE), CHO + FAP Note:*DAR determined by LC-MS; **DAR determined by HIC

FIG. 31 shows a comparison of in vitro potency of anti-HERglycoconjugate and its counterpart thiol conjugate. Cell viability wasdetermined following 72 hr exposure of the conjugates to Her2 antigenexpressing (SK-BR-3) cells (FIGS. 31A and C) or non-expressing(MDA-MB-231) cells (FIGS. 31B and D). The ADCs contained either MMAE orPEGS-Do110 linked to the glycans (“glyco”) or by conventional chemistryto hinge region cysteines (“thiol”). As shown in FIGS. 30A and C,˜2-fold lower EC₅₀ was observed for the thiol conjugates compared to theglycoconjugates, which is consistent with 2-fold higher DAR in theformer than the latter. No toxicity was observed with the Her2-cell linewith any antibody up to 100 ug/ml.

Similar trends were also observed in the cell proliferation for ADCprepared with antibodies against a tumor antigen (FAP) which is highlyexpressed by reactive stromal fibroblasts in epithelial cancersincluding colon, pancreatic and breast cancer (Teicher, B. A. (2009)Antibody-drug conjugate targets. Curr Cancer Drug Targets 9, 982-1004).These conjugates were again prepared by conjugating either aminooxy MMAEdrug-linker or maleimido MMAE drug-linker to glycans or a thiol group.Cell proliferation assays of these conjugates showed that EC₅₀ of thethiol conjugate had ˜100-fold higher potency on the CHO cellstransfected with human FAP than the same cells lacking FAP expression asdepicted in FIG. 32, which shows a comparison of in vitro potency ofanti FAP B11 glycoconjugate and thiol conjugate. Cell viability wasdetermined following exposure of the conjugates to CHO cells transfectedwith or without FAP antigen. The ADCs contained MMAE linked to theglycans (“glyco”) or by conventional chemistry to hinge region cysteines(“thiol”). Note that the ˜2-fold lower EC50 for the thiol compared tothe glycoconjugates is consistent with the relative amounts of drugdelivered per antibody assuming similar efficiencies for target bindingand internalization in antigen expressing CHO cells. In parallel, aglycoconjugate of anti FAP (B11) ADC with a DAR of 1.5 as describedpreviously was assayed and showed an ˜2-fold higher EC₅₀ than comparatorthiol conjugate (DAR 3.3).

In summary, the site-specific conjugation of the drugs through theglycans with cleavable linkers produces ADCs with toxicities and invitro efficacy that are equivalent to conventional thiol-basedconjugates, as demonstrated using different antibodies and differentdrug-linkers. Moreover, below 2 mM periodate, the level of drugconjugation correlates with the reduction of sialic acid. Increasingperiodate concentration above 2 mM produces little benefit, as expectedfrom the complete conversion of sialic acid to the oxidized form.However, under all conditions, the number of drugs per antibody wasslightly lower than the sialic acid content, indicating that some of theoxidized sialic acids may similarly not be available for coupling,either because of being buried or otherwise due to steric hindrancearising from the bulk of the drug-linker.

Example 15 In Vivo Characterization of Antibody Drug Conjugates

Efficacy of anti-HER glycoconjugates were also evaluated in a Her2+tumor cell xenograft mode and compared with thiol conjugate comparatorshaving ˜2-fold higher DAR. Nude mice were implanted with SK-OV-3 Her2+tumor cells which were allowed to establish tumors of ˜150 mm³ prior toinitiation of treatment. ADCs at 3 or 10 mg/kg doses were injectedthrough tail vein on days 38, 45, 52 and 59. There were ˜10 mice pergroup. The tumor volume of mice in different group was measured andtheir survival was recorded. The survival curve was plotted based onKaplan-Meier method.

FIG. 33 shows a comparison of in vivo efficacy of the anti-HERglycoconjugates and thiol conjugates in a Her2+ tumor cell xenograftmodel. Nude mice implanted with SK-OV-3 Her2+ tumor cells were dosedwith MMAE (FIGS. 33 A and B) and PEGS-Do110 (FIGS. 33 C and D)containing glycoconjugates or a thiol conjugate comparators with ˜2-foldhigher DAR. The tumor growth kinetics of the MMAE conjugates is shown inFIG. 33A. In this case, the glycoconjugate showed a significantly higherefficacy than the naked antibody alone (black) but less than a thiolconjugate comparator having a ˜2-fold higher DAR (green). The MMAEglycoconjugate showed significant tumor regression and a ˜20 day delayin tumor growth (FIG. 33A) and ˜2-fold increase in survival time fromfirst dose (FIG. 33B). The thiol MMAE conjugate showed near-completetumor suppression at the same dose of ADC (10 mg/kg).

The in vivo efficacy of a PEGS-Do110 glycoconjugate (“Glyco Do110’) anda thiol conjugate comparator with ˜2-fold higher DAR (“Thiol Do110”) wasalso determined in the same Her2+ tumor cell xenograft model. Bothconjugates showed lower efficacy than MMAE conjugates as describedpreviously. However, the aminooxy-PEGS-Do110 glycoconjugate (“GlycoDo110”) at 10 mg/kg showed a 15-day delay in tumor growth (FIG. 33C) and˜20 day (1.7-fold) increase in survival time following firstadministration (FIG. 33D). The thiol conjugate was more efficacious atthe same dose, showing a 2-fold increase in survival. At a lower dose (3mg/kg), the thiol conjugate showed a lesser efficacy than theglycoconjugate at 10 mg/kg. This dose corresponds to 80 umol PEGS-Do110drug per kg dose, compared to 110 umol PEGS-Do110 drug per kg dose forthe glycoconjugate.

These data demonstrate that site-specific conjugation of drugs ontosialic acid of antibody glycans yields molecules with comparable potencyas ADCs generated via thiol-based chemistry. The somewhat lower in vivoefficacy likely stems from the fewer number of drugs which are carriedby each antibody into the tumor cells by the internalization of eachantibody-bound antigen. Although we have not compared theseglycoconjugates with thiol conjugates of the same DAR, the efficacyobserved at different doses of the two ADCs representing comparablelevels of administered drug shows that the glycoconjugates havecomparable intrinsic efficacy as their thiol counterparts, indicating nodeleterious effect of conjugation at this site. Moreover, a 10 mg/kgdose of the Do110 glycoconjugate which introduced only 28% more drugprovided a 2-fold increase in survival over the thiol conjugate (at 3mg/kg), suggesting these conjugates may even provide superior efficaciesat the same DAR. Given the apparent limitation in sialic acidincorporation at native glycans, higher drug loading could be achievedby a number of different strategies including the use of branched druglinkers or the introduction of additional glycosylation sites and usingthe same method.

We claim:
 1. An isolated polynucleotide encoding a binding polypeptidecomprising a CH1 domain, or a variant thereof, wherein the CH1 domain orthe variant thereof comprises an asparagine residue at amino acidposition 114, according to Kabat numbering.
 2. A vector comprising thepolynucleotide of claim
 1. 3. A host cell comprising the polynucleotideof claim
 1. 4. A method of making a binding polypeptide comprisingexpressing the polynucleotide of claim 1 in a cell.
 5. An isolated hostcell comprising the vector of claim
 2. 6. A method of making a bindingpolypeptide comprising expressing the vector of claim 2 in a cell. 7.The isolated polynucleotide of claim 1, wherein the binding polypeptideis an antibody, or an antigen-binding fragment thereof.
 8. The isolatedpolynucleotide of claim 1, wherein the CH1 domain is an IgG1 CH1 domain.9. The isolated polynucleotide of claim 8, wherein the CH1 domain is ahuman IgG1 CH1 domain.
 10. An isolated polynucleotide encoding a bindingpolypeptide comprising a CH1 domain, or a variant thereof, wherein theCH1 domain or the variant thereof comprises: an asparagine residue atamino acid position 114, according to Kabat numbering; any amino acidresidue except proline at position 115, according to Kabat numbering;and a serine or threonine residue at position 116, according to Kabatnumbering.
 11. A vector comprising the polynucleotide of claim
 10. 12.An isolated host cell comprising the polynucleotide of claim
 10. 13. Amethod of making a binding polypeptide comprising expressing thepolynucleotide of claim 10 in a cell.
 14. An isolated host cellcomprising the vector of claim
 11. 15. A method of making a bindingpolypeptide comprising expressing the vector of claim 11 in a cell. 16.The isolated polynucleotide of claim 10, wherein the binding polypeptideis an antibody, or an antigen-binding fragment thereof.
 17. The isolatedpolynucleotide of claim 10, wherein the binding polypeptide is anantibody, or an antigen-binding fragment thereof.
 18. The isolatedpolynucleotide of claim 10, wherein the CH1 domain is an IgG1 CH1domain.
 19. The isolated polynucleotide of claim 18, wherein the CH1domain is a human IgG1 CH1 domain.